Encapsulation of pancreatic cells derived from human pluripotent stem cells

ABSTRACT

The present invention relates to methods for encapsulating pancreatic progenitors in a biocompatible semi-permeable encapsulating device. The present invention also relates to production of human insulin in a mammal in response to glucose stimulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/850,978, entitled “ENCAPSULATION OF PANCREATIC CELLS DERIVED FROMHUMAN PLURIPOTENT STEM CELLS,” filed Mar. 26, 2013, which is a divisionof U.S. patent application Ser. No. 13/188,706, entitled “ENCAPSULATIONOF PANCREATIC CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS,” filedJul. 22, 2011, which is a division of U.S. patent application Ser. No.12/618,659, entitled “ENCAPSULATION OF PANCREATIC CELLS DERIVED FROMHUMAN PLURIPOTENT STEM CELLS,” filed Nov. 13, 2009, which is anonprovisional of and which claims priority under 35 US.C. §119(e) toU.S. Provisional Patent Application No. 61/121,086, entitledENCAPSULATION OF PANCREATIC ENDODERM CELLS, filed Dec. 9, 2008 and U.S.Provisional Patent Application No. 61/114,857, entitled ENCAPSULATION OFPANCREATIC PROGENITORS DERIVED FROM HES CELLS, filed Nov. 14, 2008. Thedisclosure of each of the above-listed priority applications isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and cellbiology. In particular, the present invention relates to theencapsulation of cells derived from human embryonic stem cells and otherpluripotent human cells.

BACKGROUND OF THE INVENTION

Human embryonic stem (hES) cells and induced pluripotent stem (iPS)cells from adult differentiated cells are uniquely suited for celltherapy applications because they are pluripotent and self-renewable.Owing to the large variety of cell types that can arise indifferentiating pluripotent stem cell cultures, success in achievingefficient, directed differentiation is useful for therapeuticapplication of human pluripotent stem cells. Efficient directeddifferentiation of human pluripotent stem cells to various intermediatecell types including pancreatic lineage cells using various growth andsignaling factors and small molecules is necessary.

SUMMARY OF THE INVENTION

Embodiments described herein relate to methods of producing insulin in amammal by providing an implantable chamber into a host mammal, providinga pancreatic progenitor cell derived from human pluripotent stem cell(e.g., hES or iPS cells) to said chamber, maturing the pancreaticprogenitor cell to a mature pancreatic hormone secreting cell, whereinthe pancreatic hormone secreting cell is an insulin secreting cell whichproduces insulin in response to glucose stimulation in vivo, therebyproducing insulin in vivo in the mammal. In some embodiments, thechamber is implanted into the mammal prior to introducing the pancreaticprogenitor cell. In other embodiments, the chamber is allowed tovascularize prior to introducing the pancreatic progenitor cell. In yetother embodiments, the cell is introduced into the chamber prior toimplantation.

One embodiment relates to a method for producing insulin in a mammal,comprising: (a) providing a human PDX1-positive pancreatic progenitorcell population into an implantable semi-permeable device; (b) maturingthe cell population in said device to an islet, wherein the isletcomprises endocrine and acinar cells, and wherein the endocrine cell isat least an insulin secreting cell which produces insulin in response toglucose stimulation in vivo, thereby producing insulin in vivo to themammal.

Another embodiment relates to a cell encapsulating assembly forimplanting a cell population into a mammalian host. In one aspect, theassembly comprises a sealed periphery defining at least one chamber forencapsulating living cells. In another aspect, the assembly comprises awall means having a peripheral edge, wherein the assembly comprises afirst seal at the peripheral edge of the wall means, thereby forming theencapsulating assembly. In some aspects, the assembly comprises a secondseal which effectively reduces the chamber volume.

Another embodiment relates to a cryopreserved human pancreaticprogenitor cell population. In one aspect of the embodiment, the cellpopulation is suitable for transplantation into a mammal.

Another embodiment relates to a method of obtaining a population ofcells suitable for transplantation. In one aspect of the embodiment,cells suitable for transplantation are obtained by a method comprising:a) contacting a population of human pancreatic progenitor cells with acryopreservation solution to thereby obtain a population of cells forcryopreservation; b) decreasing the temperature of the progenitor cellsfor cryopreservation to about −196° C. to obtain cryopreserved cells;and c) increasing the temperature of the cryopreserved cells to therebyobtain a population of pancreatic progenitor cells suitable fortransplantation. In some embodiments the temperature of the progenitorcells for cryopreservation is decreased to less than 0° C., −10° C.,−20° C., −30° C., −40° C., −50° C., −60° C., −70° C., −80° C., −90° C.,−100° C., −110° C., −120° C., −130° C., −140° C., −150° C., −160° C.,−170° C., −180° C., −190° C., −200° C., −210° C., −220° C., −230° C.,−240° C., −250° C., or −260° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a dual ported encapsulation device withan internal ultrasonic weld to compartmentalize the main lumen.

FIG. 2 is a top section view of the encapsulation device shown in FIG.1.

FIG. 3 is a side view of the encapsulation device shown in FIG. 1 with across section taken through the center of the device along the internalultrasonic weld region.

FIG. 4 is a side view of the encapsulation device shown in FIG. 1 with across section taken through the center of a compartmentalized lumenalong the axis of the port.

FIG. 5 is an end view of the encapsulation device shown in FIG. 1 with across section taken through the compartmentalized lumens.

FIG. 6 is a perspective view of an encapsulation device without loadingports and containing periodic ultrasonic spot-welds to compartmentalizethe internal lumen.

FIG. 7 is a top cross section view of the encapsulation device shown inFIG. 6

FIG. 8 is a side view of the encapsulation device shown in FIG. 6 with across section taken through the center of a compartmentalized lumen.

FIG. 9 is an end view of the encapsulation device shown in FIG. 6 with across section through the compartmentalized lumens.

FIG. 10 is a perspective view of an encapsulation device without loadingports and containing periodic ultrasonic spot-welds to compartmentalizethe internal lumen. Each of the spot welds has the center removed tofacilitate vascularization.

FIG. 11 is an enlarged view of the encapsulation device shown in FIG.10.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments described herein are directed to methods of producinginsulin in vivo by implanting in a mammal human pancreatic progenitorcells derived from human embryonic stem cells in encapsulating devices,including a bio-compatible polyethylene glycol-based device and amechanical/medical device.

Unless otherwise noted, the terms used herein are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. In addition to the definitions of terms provided below,definitions of common terms in molecular biology may also be found inRieger et al., 1991 Glossary of genetics: classical and molecular, 5thEd., Berlin: Springer-Verlag; and in Current Protocols in MolecularBiology, F. M. Ausubel et al., Eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1998 Supplement). It is to be understood that as used in thespecification and in the claims, “a” or “an” can mean one or more,depending upon the context in which it is used. Thus, for example,reference to “a cell” can mean that at least one cell can be utilized.

Also, for the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

In one embodiment, hES-derived cells are encapsulated using abio-compatible polyethylene glycol (PEG). PEG-based encapsulation isdescribed in more detail in U.S. Pat. No. 7,427,415, entitledIMPLANTATION OF ENCAPSULATED BIOLOGICAL MATERIALS FOR TREATING DISEASES;U.S. Pat. No. 6,911,227, entitled GELS FOR ENCAPSULATION OF BIOLOGICALMATERIALS; and U.S. Pat. Nos. 6,911,227, 5,529,914, 5,801,033,6,258,870, entitled GELS FOR ENCAPSULATION OF BIOLOGICAL MATERIALS,which is herein incorporated by reference in their entireties.

In another embodiment, the encapsulating device is a TheraCyte device(Irvine, Calif.). TheraCyte cell encapsulation devices are furtherdescribed in U.S. Pat. Nos. 6,773,458; 6,156,305; 6,060,640; 5,964,804;5,964,261; 5,882,354; 5,807,406; 5,800,529; 5,782,912; 5,741,330;5,733,336; 5,713,888; 5,653,756; 5,593,440; 5,569,462; 5,549,675;5,545,223; 5,453,278; 5,421,923; 5,344,454; 5,314,471; 5,324,518;5,219,361; 5,100,392; and 5,011,494, which are all herein incorporatedin their entireties by reference in their entireties.

In one embodiment, methods are described for producing hES cellaggregate suspensions from a single cell suspension of pluripotent stemcell cultures or hES-derived cell cultures. The pluripotent stem cellcan be initially cultured on fibroblast feeders, or they can befeeder-free. Methods of isolating hESC and culturing such on humanfeeder cells was described in U.S. Pat. No. 7,432,104 entitled METHODSFOR THE CULTURE OF HUMAN EMBRYONIC STEM CELLS ON HUMAN FEEDER CELLS,which is herein incorporated by reference in its entirety. Variousmethods for producing hES cell aggregate suspension cultures and/orhES-derived cell aggregate suspension cultures are described in detailin U.S. application Ser. No. 12/264,760, entitled STEM CELL AGGREGATESUSPENSION COMPOSITIONS AND METHODS OF DIFFERENTIATION THEREOF, filedOct. 4, 2008, which is herein incorporated by reference in its entirety.

The differentiation culture conditions and hES-derived cell typesdescribed herein are substantially similar to that described in D'Amouret al. 2006, supra or those described in U.S. Pat. No. 7,534,608; U.S.patent application Ser. No. 11/681,687, filed Mar. 2, 2007; and Ser. No.11/773,944, filed Jul. 5, 2007, the disclosures of which areincorporated herein by reference in their entireties. D'Amour et al.describe a 5 step differentiation protocol: stage 1 (results in mostlydefinitive endoderm production), stage 2 (results in mostlyPDX1-negative foregut endoderm production), stage 3 (results in mostlyPDX1-positive foregut endoderm production), stage 4 (results in mostlypancreatic endoderm or pancreatic endocrine progenitor production) andstage 5 (results in mostly hormone expressing endocrine cellproduction).

As used herein, “definitive endoderm (DE)” refers to a multipotentendoderm lineage cell that can differentiate into cells of the gut tubeor organs derived from the gut tube. In accordance with certainembodiments, the definitive endoderm cells are mammalian cells, and in apreferred embodiment, the definitive endoderm cells are human cells. Insome embodiments, definitive endoderm cells express or fail tosignificantly express certain markers. In some embodiments, one or moremarkers selected from CER, FOZA2, SOX17, CXCR4, MIXL1, GATA4, HNF3-β,GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 are expressed indefinitive endoderm cells. In other embodiments, one or more markersselected from OCT4, α-fetoprotein (AFP), Thrombomodulin™, SPARC, SOX7and HNF4-α are not significantly expressed in definitive endoderm cells.To be clear, a definitive endoderm cell is distinguished from otherendoderm-lineage cells, such as foregut endoderm or gut endoderm orPDX1-negative foregut endoderm cells, which appreciably express HNF4-αas compared to definitive endoderm. Definitive endoderm cell populationsand methods of production thereof are also described in U.S. Pat. No.7,510,876, entitled DEFINITIVE ENDODERM, which is hereby incorporated inits entirety.

Still other embodiments relate to cell cultures termed “PDX1-negativeforegut endoderm cells” or “foregut endoderm cells” or “gut endoderm” orequivalents thereof. In some embodiments, the foregut endoderm cellsexpress SOX17, HNF1-13, HNF4-αand FOXA1 markers but do not substantiallyexpress PDX1, AFP, SOX7, SOX1. PDX1-negative foregut endoderm cellpopulations and methods of production thereof are also described in U.S.application Ser. No. 11/588,693, entitled PDX1-expressing dorsal andventral foregut endoderm, filed Oct. 27, 2006 which is incorporatedherein by reference in its entirety. Again, gut endoderm appreciablyexpresses HNF4-α as compared to the definitive endoderm cells, or Stage1 cells; see Examples below.

Other embodiments described herein relate to cell cultures of“PDX1-positive, dorsally-biased, foregut endoderm cells”, “PDX1-positiveforegut endoderm cells”, or “PDX1-positive endoderm” or equivalentsthereof. In some embodiments, the PDX1-positive foregut endoderm cellsexpress PDX1, HNF6, SOX 9 and PROX 1 markers but do not substantiallyexpress NKX6.1, PTF1A, CPA, cMYC, SOX17, HNF1B or HNF4alpha.PDX1-positive foregut endoderm cell populations and methods ofproduction thereof are also described in U.S. application Ser. No.11/588,693, entitled PDX1-expressing dorsal and ventral foregutendoderm, filed Oct. 27, 2006, which is incorporated herein by referencein its entirety.

Other embodiments described herein relate to cell cultures of“pancreatic progenitors”, “PDX1-positive pancreatic endoderm cells,”“PDX1-positive pancreatic progenitor,” “pancreatic epithelium”, “PE” orequivalents thereof. PDX1-positive pancreatic progenitor cells aremultipotent and can give rise to various cells in the pancreas includingbut not limited to acinar, duct and endocrine cells. In someembodiments, the PDX1-positive pancreatic progenitor cells expressincreased levels of PDX1 and NKX6.1 as compared to non pre-pancreaticendoderm cells which do not appreciably express these markers.PDX1-positive pancreatic progenitor cells also express low to no levelsof PTF1A, CPA, cMYC, NGN3, PAX4, ARX and NKX2.2, INS, GCG, GHRL, SST,and PP.

Alternatively, other embodiments relate to cell cultures of“PDX1-positive pancreatic endoderm tip cells,” or equivalents thereof.In some embodiments, the PDX1-positive pancreatic endoderm tip cellsexpress increased levels of PDX1 and NKX6.1 similar to PDX1-positivepancreatic progenitor cells, but unlike PDX1-positive pancreaticprogenitor cells, PDX1-positive pancreatic endoderm tip cellsadditionally express increased levels of PTF1A, CPA and cMYC.PDX1-positive pancreatic endoderm tip cells also express low to nolevels of NGN3, PAX4, ARX and NKX2.2, INS, GCG, GHRL, SST, and PP.

Other embodiments relate to cell cultures of “pancreatic endocrineprecursor cells,” “pancreatic endocrine progenitor cells” or equivalentsthereof. Pancreatic endocrine progenitor cells are multipotent and giverise to mature endocrine cells including alpha, beta, delta and PPcells. In some embodiments, the pancreatic endocrine progenitor cellsexpress increased levels of NGN3, PAX4, ARX and NKX2.2 as compared toother non-endocrine progenitor cell types. Pancreatic progenitor cellsalso express low to no levels of INS, GCG, GHRL, SST, and PP.

Still other embodiments relate to cell cultures of “pancreatic endocrinecells,” “pancreatic hormone secreting cells”, “pancreatic islethormone-expressing cell,” or equivalents thereof refer to a cell, whichhas been derived from a pluripotent cell in vitro, e.g. alpha, beta,delta and/or PP cells or combinations thereof. The endocrine cells canbe poly-hormonal or singly-hormonal, e.g. expressing insulin, glucagon,ghrelin, somatostatin and pancreatic polypeptide or combinationsthereof. The endocrine cells can therefore express one or morepancreatic hormones, which have at least some of the functions of ahuman pancreatic islet cell. Pancreatic islet hormone-expressing cellscan be mature or immature. Immature pancreatic islet hormone-expressingcells can be distinguished from mature pancreatic islethormone-expressing cells based on the differential expression of certainmarkers, or based on their functional capabilities, e.g., glucoseresponsiveness in vitro or in vivo. Pancreatic endocrine cells alsoexpress low to no levels of NGN3, PAX 4, ARX and NKX2.2.

Most of above cell types are epithelialized as compared to mesenchymaldefinitive endoderm cells. In some embodiments, the pancreatic endodermcells express one or more markers selected from Table 3 and/or one ormore markers selected from Table 4 of related U.S. application Ser. No.11/588,693 entitled PDX1 EXPRESSING DOSAL AND VENTRAL FOREGUT ENDODERM,filed Oct. 27, 2006, and also U.S. application Ser. No. 11/115,868,entitled PDX1-expressing endoderm, filed Apr. 26, 2005, which are herebyincorporated herein by reference in their entireties.

In certain embodiments, the terms “enriched”, “isolated”, “separated”,“sorted”, “purified” or purifying by depleting or equivalents thereofrefer to a cell culture or a cell population or cell sample thatcontains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the desiredcell lineage or a desired cell having a certain cell phenotype, e.g.,expressing a certain cell marker or not expressing a certain cell markergene characteristic of that cell phenotype. Methods for purifying,enriching, isolating, separating, sorting, and/or depleting endodermlineage cells derived from hES cells are also described in U.S.application Ser. No. 12/107,020, entitled METHODS FOR PURIFYING ENDODERMAND PANCREATIC ENDODERM CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS,filed Apr. 21, 2008 which is incorporated herein by reference in itsentirety.

As used herein, the term “contacting” (i.e., contacting a cell e.g., adifferentiable cell, with a compound) is intended to include incubatingthe compound and the cell together in vitro (e.g., adding the compoundto cells in culture). The term “contacting” is not intended to includethe in vivo exposure of cells to a defined cell medium comprising anErbB3 ligand, and optionally, a member of the TGF-β family, that mayoccur naturally in a subject (i.e., exposure that may occur as a resultof a natural physiological process). The step of contacting the cellwith a defined cell medium comprising an ErbB3 ligand, and optionally, amember of the TGF-β family, can be conducted in any suitable manner. Forexample, the cells may be treated in adherent culture, or in suspensionculture. It is understood that the cells contacted with the definedmedium can be further treated with a cell differentiation environment tostabilize the cells, or to differentiate the cells.

As used herein, the term “differentiate” refers to the production of acell type that is more differentiated than the cell type from which itis derived. In some embodiments, the term “differentiate” means toproduce a cell that has fewer fate choices than the cell from which itwas derived. The term therefore encompasses cell types that arepartially and terminally differentiated. Differentiated cells derivedfrom hES cells are generally referred to as hES-derived cells orhES-derived cell aggregate cultures, or hES-derived single cellsuspensions, or hES-derived cell adherent cultures and the like.

As used herein, the term “differentiable cell” is used to describe acell or population of cells that can differentiate into at leastpartially mature cells, or that can participate in the differentiationof cells, e.g., fuse with other cells, that can differentiate into atleast partially mature cells. As used herein, “partially mature cells”,“progenitor cells”, “immature cells”, “precursor cells”, “multipotentcells” or equivalents thereof include those cells which are notterminally differentiated, e.g., definitive endoderm cells,PDX1-negative foregut endoderm cells, PDX1-positive pancreatic endodermcells which further include PDX1-positive pre-pancreatic endoderm cellsand PDX1-positive pancreatic endoderm tip cells. All are cells thatexhibit at least one characteristic of the phenotype, such as morphologyor protein expression, of a mature cell from the same organ or tissuebut can further differentiate into at least one other cell type. Forexample, a normal, mature hepatocyte typically expresses such proteinsas albumin, fibrinogen, α-1-antitrypsin, prothrombin clotting factors,transferrin, and detoxification enzymes such as the cytochrome P-450s,among others. Thus, as used herein, a “partially mature hepatocyte” mayexpress albumin or another one or more proteins, or begin to take theappearance or function of a normal, mature hepatocyte.

As used herein, the term “substantially” refers to a great extent ordegree, e.g. “substantially similar” in context would be used todescribe one method which is to great extent or degree similar toanother method. However, as used herein, the term “substantially free”,e.g., “substantially free” or “substantially free from contaminants,” or“substantially free of serum” or “substantially free of insulin orinsulin like growth factor” or equivalents thereof, is meant that thesolution, media, supplement, excipient and the like, is at least 98%, orat least 98.5%, or at least 99%, or at least 99.5%, or at least 100%free of serum, contaminants or equivalent thereof. In one embodiment, adefined culture media contains no serum, or is 100% serum-free, or issubstantially free of serum. Conversely, as used herein, the term“substantially similar” or equivalents thereof is meant that thecomposition, process, method, solution, media, supplement, excipient andthe like is meant that the process, method, solution etc., is at least50%, 55%, 60%, 65%, 70%, 75%, 80%, at least 85%, at least 90%, at least95%, or at least 99% similar to that previously described in thespecification herein, or in a previously described process or methodincorporated herein in its entirety.

Also, as used herein, in connection with the composition of a cellpopulation, the term “essentially” or “substantially” meanspredominantly or mainly. In some embodiments these terms mean at least85% of the cells in a cell population, at least 86% of the cells in acell population, at least 87% of the cells in a cell population, atleast 88% of the cells in a cell population, at least 89% of the cellsin a cell population, at least 90% of the cells in a cell population, atleast 91% of the cells in a cell population, at least 92% of the cellsin a cell population, at least 93% of the cells in a cell population, atleast 94% of the cells in a cell population, at least 95% of the cellsin a cell population, at least 96% of the cells in a cell population, atleast 97% of the cells in a cell population, at least 98% of the cellsin a cell population, or at least 99% of the cells in a cell population.In other embodiments, the terms or phrases “essentially free of” and“substantially free of” refer to a de minimus or a reduced amount of acomponent or cell present in any cell culture, e.g., pancreaticprogenitors as described herein are “essentially or substantiallyhomogenous”, “essentially or substantially homo-cellular”, “essentiallyhES cells”, “essentially or substantially definitive endoderm cells”,“essentially or substantially foregut endoderm cells”, “essentially orsubstantially gut endoderm cells”, “essentially or substantiallyPDX1-negative foregut endoderm cells”, “essentially or substantiallyPDX1-positive pre-pancreatic endoderm cells”, “essentially orsubstantially PDX1-positive pancreatic progenitor cells”, “essentiallyor substantially pancreatic epithelial cells”, “essentially orsubstantially PDX1-positive pancreatic endoderm tip cells”, “essentiallyor substantially pancreatic endocrine precursor cells”, “essentially orsubstantially pancreatic endocrine cells” and the like. The terms,“essentially” and “substantially” can also mean that at least 50%, 55%,60%, 65%, 70%, 75%, 80%, at least 85%, at least 90%, at least 95%, or atleast 99% that cell (definitive endoderm; PDX1-negative foregutendoderm; PDX1-positive pre-pancreatic endoderm; PDX1-positivepancreatic progenitor cells; PDX1-positive pancreatic tip cells;endocrine precursor cells, and endocrine hormone-secreting cells)

As used herein, the term “effective amount” or equivalents thereof of acompound refers to that concentration of the compound that is sufficientin the presence of the remaining components of the defined medium toeffect the stabilization of the differentiable cell in culture forgreater than one month in the absence of a feeder cell and in theabsence of serum or serum replacement. This concentration is readilydetermined by one of ordinary skill in the art.

As used herein, the term “express” refers to the transcription of apolynucleotide or translation of a polypeptide in a cell, such thatlevels of the molecule are measurably higher in a cell that expressesthe molecule than they are in a cell that does not express the molecule.Methods to measure the expression of a molecule are well known to thoseof ordinary skill in the art, and include without limitation, Northernblotting, RT-PCR, in situ hybridization, Western blotting, andimmunostaining.

As used herein when referring to a cell, cell line, cell culture orpopulation of cells, the term “isolated” refers to being substantiallyseparated from the natural source of the cells such that the cell, cellline, cell culture, or population of cells are capable of being culturedin vitro. In addition, the term “isolating” is used to refer to thephysical selection of one or more cells out of a group of two or morecells, wherein the cells are selected based on cell morphology and/orthe expression of various markers.

As used herein, the term “preserving cells” means maintaining cells in aviable state for a period of time before transplantation. The period oftime may be 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24hours, 2 days, 4 days, 5 days, 1 week, 2 weeks, 4 weeks, 1 month, 2months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 4years, 6 years, 8 years, 10 years, 12 years, 14 years, 16 years, 18years, 20 years, 22 years, 24 years, 30 years, 35 years, 40 years, 45years, or 100 years or any period of time between any times provided inthis range.

Differentiable cells, as used herein, may be pluripotent, multipotent,oligopotent or even unipotent. In certain embodiments, thedifferentiable cells are pluripotent differentiable cells. In morespecific embodiments, the pluripotent differentiable cells are selectedfrom the group consisting of embryonic stem cells, ICM/epiblast cells,primitive ectoderm cells, primordial germ cells, and teratocarcinomacells. In some embodiments, the differentiable cells are derived from apreimplantation embryo. In one particular embodiment, the differentiablecells are mammalian embryonic stem cells. In a more particularembodiment, the differentiable cells are human embryonic stem cells.

The cell types that differentiate from differentiable cells have severaluses in various fields of research and development including but notlimited to drug discovery, drug development and testing, toxicology,production of cells for therapeutic purposes as well as basic scienceresearch. These cell types express molecules that are of interest in awide range of research fields. These include the molecules known to berequired for the function of the various cell types as described instandard reference texts. These molecules include, but are not limitedto, cytokines, growth factors, cytokine receptors, extracellular matrix,transcription factors, secreted polypeptides and other molecules, andgrowth factor receptors.

It is contemplated that differentiable cells can be differentiatedthrough contact with a cell differentiation environment. As used herein,the term “cell differentiation environment” refers to a cell culturecondition wherein the differentiable cells are induced to differentiate,or are induced to become a human cell culture enriched in differentiatedcells. Preferably, the differentiated cell lineage induced by the growthfactor will be homogeneous in nature. The term “homogeneous,” refers toa population that contains more than approximately 50%, 60%, 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% of the desired cell lineage.

A cell differentiating medium or environment may be utilized topartially, terminally, or reversibly differentiate the differentiablecells described herein. In accordance with the embodiments describedherein, the medium of the cell differentiation environment may contain avariety of components including, for example, KODMEM medium (KnockoutDulbecco's Modified Eagle's Medium), DMEM, Ham's F12 medium, FBS (fetalbovine serum), FGF2 (fibroblast growth factor 2), KSR or hLIF (humanleukemia inhibitory factor). The cell differentiation environment canalso contain supplements such as L-Glutamine, NEAA (non-essential aminoacids), P/S (penicillin/streptomycin), N2, B27 and β-mercaptoethanol(β-ME). It is contemplated that additional factors may be added to thecell differentiation environment, including, but not limited to,fibronectin, laminin, heparin, heparin sulfate, retinoic acid, membersof the epidermal growth factor family (EGFs), members of the fibroblastgrowth factor family (FGFs) including FGF2, FGF7, FGF8, and/or FGF10,members of the platelet derived growth factor family (PDGFs),transforming growth factor (TGF)/bone morphogenetic protein (BMP)/growthand differentiation factor (GDF) factor family antagonists including butnot limited to noggin, follistatin, chordin, gremlin, cerberus/DANfamily proteins, ventropin, high dose activin, and amnionless orvariants or functional fragments thereof. TGF/BMP/GDF antagonists couldalso be added in the form of TGF/BMP/GDF receptor-Fc chimeras. Otherfactors that may be added include molecules that can activate orinactivate signaling through Notch receptor family, including but notlimited to proteins of the Delta-like and Jagged families as well asinhibitors of Notch processing or cleavage, or variants or functionalfragments thereof. Other growth factors may include members of theinsulin like growth factor family (IGF), insulin, the wingless related(WNT) factor family, and the hedgehog factor family or variants orfunctional fragments thereof. Additional factors may be added to promotemesendoderm stem/progenitor, endoderm stem/progenitor, mesodermstem/progenitor, or definitive endoderm stem/progenitor proliferationand survival as well as survival and differentiation of derivatives ofthese progenitors.

The progression of the differentiable cells to the desired cell lineage,or its maintenance in an undifferentiated state can be monitored byquantitating expression of marker genes characteristic of the desiredcell lineage as well as the lack of expression of marker genescharacteristic of differentiable cell types. One method of quantitatinggene expression of such marker genes is through the use of quantitativePCR (Q-PCR). Methods of performing Q-PCR are well known in the art.Other methods that are known in the art can also be used to quantitatemarker gene expression. Marker gene expression can be detected by usingantibodies specific for the marker gene of interest.

Embodiments described herein also contemplate differentiable cells fromany source within an animal, provided the cells are differentiable asdefined herein. For example, differentiable cells may be harvested fromembryos, or any primordial germ layer therein, from placental or choriontissue, or from more mature tissue such as adult stem cells including,but not limited to adipose, bone marrow, nervous tissue, mammary tissue,liver tissue, pancreas, epithelial, respiratory, gonadal and muscletissue. In specific embodiments, the differentiable cells are embryonicstem cells. In other specific embodiments, the differentiable cells areadult stem cells. In still other specific embodiments, the stem cellsare placental- or chorionic-derived stem cells.

Other embodiments contemplate using differentiable cells from any animalcapable of generating differentiable cells. The animals from which thedifferentiable cells are harvested may be vertebrate or invertebrate,mammalian or non-mammalian, human or non-human. Examples of animalsources include, but are not limited to, primates, rodents, canines,felines, equines, bovines and porcines.

Some embodiments contemplate using induced pluripotent stem (iPS) cells,which are pluripotent stem cells derived from a non-pluripotent cell.See Zhou et al. (2009), Cell Stem Cell 4: 381-384; Yu et al., (2009)Science 324(5928):797-801, Epub Mar. 26, 2009; Yu et al. (2007) Science318(5858):1917-20, Epub Nov. 20, 2007; Takahashi et al., (2007) Cell,131:861-72; and Takahashi K. and Yamanaka S. (2006), Cell 126:663-76,which are herein incorporated by reference in their entireties. Theanimals from which the non-pluripotent cells are harvested may bevertebrate or invertebrate, mammalian or non-mammalian, human ornon-human. Examples of animal sources include, but are not limited to,primates, rodents, canines, felines, equines, bovines and porcines.

The differentiable cells described herein can be derived using anymethod known to those of skill in the art. For example, humanpluripotent cells can be produced using de-differentiation and nucleartransfer methods. Additionally, the human ICM/epiblast cell or theprimitive ectoderm cell used herein is derived in vivo or in vitro.Primitive ectodermal cells may be generated in adherent culture or ascell aggregates in suspension culture, as described in WO 99/53021.Furthermore, the human pluripotent cells can be passaged using anymethod known to those of skill in the art, including, manual passagingmethods, and bulk passaging methods such as enzymatic or non-enzymaticpassaging.

In certain embodiment, when ES cells are utilized, the embryonic stemcells have a normal karyotype, while in other embodiments, the embryonicstem cells have an abnormal karyotype. In one embodiment, a majority ofthe embryonic stem cells have a normal karyotype. It is contemplatedthat greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greaterthan 95% of metaphases examined will display a normal karyotype.

Storing Cells for Encapsulation and Transplantation

Some embodiments relate to methods for cyropreserving cells which havebeen cultured and/or differentiated in vitro Such storage would allowbanking, quality control, and other desired procedures andmanipulations, either in connection with in vitro analysis orimplantation in vivo. Methods for cell storage prior to transplantationinclude preserving the tissue by freezing cells (cryopreservation); orby refrigerating the cells at above freezing temperatures (hibernation).See Chanaud et al. 1987 Neurosci Lett 82: 127-133; Collier et al. (1987)436: 363-366; and Sauer et al. 1991 Neurology and Neuroscience 2:123-135; Gage et al. 1985 Neurosci Lett 60: 133-137, the disclosures ofwhich are herein incorporated by reference in their entireties. Althoughhibernation has been reported to increase rates of graft survival andfunction as compared to cryopreserved tissue, cells may not be capableof long term maintenance under such conditions without jeopardizing cellviability during the hibernation period.

As used herein, a “cell suspension” or equivalents thereof refers tocell aggregates and/or clusters and/or spheres that are contacted with amedium. Such cell suspensions are described in detail in U.S.application Ser. No. 12/264,760, entitled Stem cell Aggregate SuspensionCompositions and Methods of Differentiation Thereof, filed on Nov. 8,2008, the disclosure of which is herein incorporated by reference in itsentirety.

As used herein, “adapted cell suspension” or cell suspension cultures orequivalents thereof includes a cell suspension that has been storedabove freezing, preferably at 4° C., in hibernation medium for about 1hour and up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or up to 30 days.

As used herein, a cell suitable for transplantation refers to a cell ora population of cells sufficiently viable and/or functional for in vivotreatment of a metabolic disorder. For example, diabetes, or one or moresymptoms thereof, can be ameliorated or reduced for a period of timefollowing implantation of a cell suitable for transplantation into asubject suffering from diabetes. In one preferred embodiment, a cell orcell population suitable for transplantation is a pancreatic progenitorcell or population, or a PDX1-positive pancreatic progenitor cell orpopulation, or an endocrine precursor cell or population, or a poly orsingly-hormonal endocrine cell and/or any combination of cell orpopulations of cells, or even purified or enriched cells or populationsof cells thereof. Cells suitable for the embodiments described hereinare further described in detail in U.S. Pat. No. 7,534,608 thedisclosure of which is herein incorporated by reference in its entirety.

As used herein the term “storing” or equivalents thereof refers toholding or maintaining cells either above or below freezing. The term isalso meant to include maintaining cells prior to use in transplantationin a subject.

As used herein the term “cryopreservation” or equivalents thereof refersto preservation of cells at temperatures below freezing.

As used herein the term “hibernation” or equivalents thereof refers topreservation of cells at temperatures above freezing and sufficientlybelow normal physiological temperature such that one or more normalcellular physiological processes are decreased or halted. In oneembodiment, preferred hibernation temperatures range between 0 and 4°C., preferably about 4° C. Hibernation medium as used herein includesany medium which lacks a cryopreservative and is physiologicallycompatible for storage of a cell at above freezing temperatures,preferably about 4° C.

Hibernation Conditions

Hibernation temperatures typically range from between 0 and 5° C.,preferably about 4° C. Numerous types of media can be used ashibernation media in conjunction with the instant methods. Prior artmethods for freezing and hibernating cells utilize complex mediacomprising buffers and added protein, sometimes including entirelyundefined components, such as serum. However, to minimize toxicity andimmunogenicity such additives are not desirable for transplantation intohumans. In preferred embodiments, hibernation media is free of addedCa⁺. In certain embodiments, medium for hibernating cells is free ofadded protein and/or free of a buffer. A preferred hibernation mediumincludes or consists of minimal amounts of glucose or moderate amountsof glucose in a saline solution, e.g., either no additional glucose orbetween about 0.1%-0.9% glucose in saline. In preferred embodiments, thehibernation medium includes or consists of about 0.1-0.5% glucose. In amore preferred embodiment, the medium includes or consists of about 0.2%glucose. In preferred embodiments, the hibernation medium includes orconsists of a very small percentage (vol/vol) of NaCl, e.g., about0.1-1% NaCl, preferably about 0.5-0.9% NaCl. In certain embodiments,more complex media can be used, e.g., Hank's balanced salt solution,Dulbecco's minimal essential medium, or Eagle's modified minimalessential medium. In certain embodiments it may be desirable tosupplement the chosen hibernation medium with additives, for example,added protein (e.g., mammalian serum protein or whole serum (preferablyheat inactivated)) buffers (e.g., phosphate buffers, HEPES, or the like)antioxidants, growth factors, KCl (e.g., at about 30 mM), lactate (e.g.,at about 20 mM), pyruvate, MgCl₂ (e.g., at about 2-3 mM), sorbitol(e.g., at about 300 mM) or other additives as are well known in the art.

In certain embodiments, the cells are hibernated at about 0-5° C.,preferably about 4° C. In certain embodiments, cells are maintained atabout 4° C. in hibernation medium prior to freezing or use. In otherembodiments, the cells are maintained at about 4° C. in hibernationmedium post freezing. In still other embodiments, the cells aremaintained at about 4° C. in hibernation medium without freezing. Incertain embodiments, the cells are maintained in hibernation medium atabout 4° C. for at least about 1 hour and up to about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or up to 30 days prior to freezing, post freezing or prior to use intransplantation. In other embodiments, the cells are maintained inhibernation medium at about 4° C. for at least about 12-72 hours priorto freezing, post freezing or prior to use in transplantation. Incertain embodiments the cells are maintained at 4° C. in hibernationmedium for at least about 24 hours prior to freezing, post freezing orprior to use in transplantation. In a more preferred embodiment, thecells are maintained in hibernation medium from at least about 36-48hours at about 4° C. prior to freezing, post freezing or prior to use.

Cryopreservation Conditions

In some embodiments cells are cryopreserved using a cryopreservationsolution. A cryopreservation solution or medium includes a solutionwhich contains a cryopreservative, i.e., a compound which protects cellsagainst intracellular and/or cell membrane damage as the cells arefrozen or thawed. A cryopreservative is identified by enhanced viabilityand/or functionality of cells in contact with the cryopreservative whencompared with cells which are similarly frozen or thawed in the absenceof the cryopreservative. Any cryopreservative can be used in conjunctionwith the instant methods and the term is meant to encompass bothintracellular and extracellular cryopreservatives.

Any cryopreservative known in the art can be used in a cryopreservativesolution. In certain embodiments, cryopreservation solutions includeintracellular cryopreservatives including but not limited todimethylsulfoxide (DMSO), various diols and triols (e.g., ethyleneglycol, propylene glycol, butanediol and triol and glycerol), as well asvarious amides (e.g., formamide and acetamide); and extracellularcryopreservatives including but not limited to phosphomono andphosphodiester catabolites of phosphoglycerides, polyvinylpyrrolidone,or methylcellulose (e.g., at least 0.1%) can also be used alone or incombination with any of the intracellular cryopreservatives.

In preferred embodiments, DMSO is used as the cryopreservative. DMSO canbe used at a wide range of concentrations, e.g., about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15% ormore. In more preferred embodiments the concentration of DMSO rangesfrom about 6% to about 12%. In particularly preferred embodiments theconcentration of DMSO is about 10%.

In certain embodiments, the cryopreservative is added to the cells in astepwise manner in order to gradually increase the concentration of thecryopreservative until the desired final concentration ofcryopreservative is achieved. In certain embodiments, the cells arecontacted with a cryopreservation solution containing thecryopreservative at the desired final concentration or thecryopreservative is added directly to the base medium without a gradualincrease in concentration.

The cryopreservation solution includes the cryopreservative in anappropriate base medium. Any type of media can be used for this purpose.In preferred embodiments, the base medium to which the cryopreservativeis added is free of added Ca⁺ In certain embodiments the medium to whichthe cryopreservative is added is free of added protein and/or free of abuffer. In other embodiments, the base medium (e.g. DMEM or DMEM/F12) towhich the cryopreservative is added includes or consists of about0.1-0.5% glucose or no or low glucose. In some aspects of thisembodiment, the base medium (e.g. DMEM or DMEM/F12) to which thecryopreservative is added includes or consists of about 0.5-0.9% NaCl.In preferred embodiments, the base medium to which the cryopreservativeis added includes or consists of very low to no glucose and about0.5-0.9% NaCl. In another preferred embodiment, the base medium to whichthe cryopreservative is added includes or consists of about 0.1 to 0.2%glucose. In some aspects of this embodiment, the base medium to whichthe cryopreservative is added includes or consists of about 0.5-0.9%NaCl.

In certain embodiments the cryopreservation solution can also containadded protein, for example, serum, e.g., fetal calf serum or humanserum, or a serum protein, e.g., albumin or knockout serum replacement.In other embodiments, the cryopreservative can also contain otheradditives, such as those described above for inclusion in hibernationmedia, for example, antioxidants, growth factors, KCl (e.g., at about 30mM), lactate (e.g., at about 20 mM), pyruvate, MgCl₂ (e.g., at about 2-3mM), sorbitol (e.g., to an osmolarity of about 300 mM) or otheradditives as are well known in the art.

Once the cells are suspended in cryopreservation solution, thetemperature of the cells is reduced in a controlled manner. In coolingthe cells to below freezing, the reduction in temperature preferablyoccurs slowly to allow the cells to establish an equilibrium between theintracellular and extracellular concentration of cryopreservative suchthat intracellular ice crystal formation is inhibited. In someembodiments, the rate of cooling is preferably fast enough to protectthe cells from excess water loss and the toxic effects ofcryopreservatives. The cells can then be cryopreserved at a temperatureof between −20° C. and about −250° C. Preferably, the cells are storedbelow −90° C. to minimize the risk of ice recrystalization. Inparticularly preferred embodiments, the cells are cryopreserved inliquid nitrogen at about −196° C. Alternatively, controlled freezing maybe accomplished with the aid of commercially available electronicallycontrolled freezer equipment.

Thawing Conditions

After cryopreservation, the cells can be thawed through any availablemethod. In a preferred embodiment, the cells are thawed rapidly, e.g.,by quick immersion in liquid at 37° C. Once the cells are thawed,dilution of the cryopreservative is accomplished by addition of adilution medium.

Any media can be used for diluting the cryopreservation solution whichis in contact with the thawed cells. For example, any of the medialisted above for use in hibernating cells, or for growth anddifferentiation of cells, can be used for diluting the cryopreservationsolution. Other media are also appropriate, for example, Hank's balancedsalt solution (preferably without Ca++), DMEM containing media with noglucose or minimal to low amounts of glucose. Additives, e.g., as listedabove for inclusion in hibernation or freezing media can also be used inmedia for dilution. Exemplary additives include, for example, buffers(e.g., phosphate buffers, HEPES, or the like) antioxidants, growthfactors, KCl (e.g., at about 30 mM), lactate (e.g., at about 20 mM),pyruvate, MgCl₂ (e.g., at about 2-3 mM), sorbitol (e.g., to anosmolarity of about 300 mM) or others additives as are well known in theart. Another suitable additive includes DNase (e.g., commerciallyavailable from Genentech, Incorporated as PULMOZYMEOR). The medium whichis used for diluting the cryopreservation solution can, optionally,contain added protein, e.g., added protein (e.g., mammalian serum(preferably heat inactivated) or a serum protein such as albumin. Inother embodiments, the medium contains no added protein and/or no addedbuffer.

After dilution of the cryopreservative, the cells can then be allowed tosettle or a pellet of cells can be formed under centrifugal force inorder to remove as much of the cryopreservation solution from the cellsas possible. The cells can then be washed in medium which does notcontain a cryopreservative. It may be preferable for the cells to remainat room temperature after the addition of the wash media and prior toletting the cells settle or form a pellet under centrifugal force. Inpreferred embodiments, the cells remain at room temperature for about10, 15, 20, 30 minutes prior to the second centrifugation. Any mediumknown in the art can be used to wash the cells, for example, any of thehibernation or dilution media set forth above can be used.

After thawing and washing, cells are cultured at 37° C. for varyinglengths of time to allow recovery prior to transplantation. Cells can becultured in any culture medium, preferably in medium appropriate totheir stage of differentiation. During this time some cell may deathoccur.

For use in transplantation, cells should be suspended in a final mediumwhich is suitable for administration to a subject. Transplantation ofcells is substantially similar to that described in U.S. Pat. No.7,534,608, which is herein incorporated by reference in its entirety.

In addition, the thawed cells may be maintained in hibernation medium asdescribed above at between 0 and 37° C., preferably about 4° C. for upto 1 hour and up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or up to 30 days prior to usein transplantation without a significant loss in viability. In someembodiments, no statistically significant loss in cell viability occurs.

Determining Viability of Recovered Cells

After storage, it may be desirable to assay the viability and/orfunctionality of the cells prior to transplantation to confirm theirsuitability for use, e.g., in transplantation. This can be accomplishedusing a variety of methods known in the art. For example, the cells canbe stained using vital stains, such as, e.g., trypan blue or ethidiumbromide or acridine orange. In certain embodiments, a population ofcells suitable for transplantation is at least between about 50-100%viable. In preferred embodiments, a population of cells suitable fortransplantation is at least about 50%, is at least about 55%, is atleast about 60%, is at least about 65%, is at least about 70%, is atleast about 75%, is at least about 80%, is at least about 85%, is atleast about 90%, is at least about 95%, is at least about 96%, is atleast about 97%, is at least about 98%, is at least about 99%, viable.In particularly preferred embodiments, such a population of cells is atleast about 85% viable.

In other embodiments, the morphometric characteristics of the cells canbe determined as a measure of the suitability of cells for use intransplantation. In preferred embodiments, the morphology of cells whichhave been stored using the instant methods and are suitable fortransplantation does not differ (e.g., statistically significant) fromthat of fresh cells. In preferred embodiments, the in vivo morphology ofcells which have been stored using the instant methods and are suitablefor transplantation does not differ (e.g., statistically significant)from that of fresh cells.

In the case of cell clusters, cell mass can be quantitated before andafter cell freeze/thaw and recovery. In one embodiment, cell clusterscultured in suspension can be manipulated to pack in closely. The areaoccupied by the clusters can then be photographed and measured. Bycomparing the areas occupied by cells before and after freeze/thaw andrecovery, a value for percent recovery can be determined.

Cells which have been stored can also be assayed for the presence ofcertain hES and/or pancreatic progenitor or hormone secreting cellmarkers to determine if they are suitable for use in transplantationThis method has been described in detail in the above in Kroon et al.2008, supra or in U.S. Pat. No. 7,534,608, which are herein incorporatedby reference in its entireties.

Additionally, or alternatively, the cells can be tested for theirfunctionality, e.g. as discussed in Kroon et al. 2008, supra or in U.S.Pat. No. 7,534,608, which are herein incorporated by reference in itsentireties.

Encapsulation Devices

One embodiment described herein relates to encapsulation devices. Suchdevices can be implanted into a mammal to treat a variety of diseasesand disorders. In preferred embodiments, the device comprises abiocompatible, immuno-isolating device that is capable of whollyencapsulating a therapeutically biologically active agent and/or cellstherein. For example, such devices can house therapeutically effectivequantities of cells within a semi-permeable membrane having a pore sizesuch that oxygen and other molecules important to cell survival andfunction can move through the semi-permeable membrane but the cells ofthe immune system cannot permeate or traverse through the pores.Similarly, such devices can contain therapeutically effective quantitiesof a biologically active agent, e.g., an angiogenic factor, a growthfactor, a hormone and the like.

The devices described herein can be employed for treating pathologiesrequiring a continuous supply of biologically active substances to theorganism. Such devices are, for example, can also be referred to as,bioartificial organs, which contain homogenous or heterogenous mixturesof biologically active agents and/or cells, or cells producing one ormore biologically active substances of interest. Ideally, thebiologically active agents and/or cells are wholly encapsulated orenclosed in at least one internal space or are encapsulation chambers,which are bounded by at least one or more semi-permeable membranes. Sucha semi-permeable membrane should allow the encapsulated biologicallyactive substance of interest to pass (e.g., insulin, glucagon,pancreatic polypeptide and the like), making the active substanceavailable to the target cells outside the device and in the patient'sbody. In a preferred embodiment, the semi-permeable membrane allowsnutrients naturally present in the subject to pass through the membraneto provide essential nutrients to the encapsulated cells. At the sametime, such a semi-permeable membrane prohibits or prevents the patient'scells, more particularly to the immune system cells, from passingthrough and into the device and harming the encapsulated cells in thedevice. For example, in the case of diabetes, this approach can allowglucose and oxygen to stimulate insulin-producing cells to releaseinsulin as required by the body in real time while preventing immunesystem cells from recognizing and destroying the implanted cells. In apreferred embodiment, the semi-permeable membrane prohibits theimplanted cells from escaping encapsulation.

Preferred devices may have certain characteristics which are desirablebut are not limited to one or a combination of the following: i)comprised of a biocompatible material that functions under physiologicconditions, including pH and temperature; examples include, but are notlimited to, anisotropic materials, polysulfone (PSF), nano-fiber mats,polyimide, tetrafluoroethylene/polytetrafluoroethylene (PTFE; also knownas Teflon®), ePTFE (expanded polytetrafluoroethylene),polyacrylonitrile, polyethersulfone, acrylic resin, cellulose acetate,cellulose nitrate, polyamide, as well as hydroxylpropyl methyl cellulose(HPMC) membranes; ii) releases no toxic compounds harming thebiologically active agent and/or cells encapsulated inside the device;iii) promotes secretion or release of a biologically active agent ormacromolecule across the device; iv) promotes rapid kinetics ofmacromolecule diffusion; v) promotes long-term stability of theencapsulated cells; vi) promotes vascularization; vii) comprised ofmembranes or housing structure that is chemically inert; viii) providesstable mechanical properties; ix) maintains structure/housing integrity(e.g., prevents unintended leakage of toxic or harmful agents and/orcells); x) is refillable and/or flushable; xi) is mechanicallyexpandable; xii) contains no ports or at least one, two, three or moreports; xiii) provides a means for immuno-isolating the transplantedcells from the host tissue; xiv) is easy to fabricate and manufacture;and xv) can be sterilized.

The embodiments of the encapsulation devices described herein are in notintended to be limited to certain device size, shape, design, volumecapacity, and/or materials used to make the encapsulation devices, solong as one or more of the above elements are achieved.

Device Designs

In one embodiment, the encapsulated device is improved by creating oneor more compartments in the device, other than that created by sealingor welding the device around the periphery or edges to prevent leakageof the cells and/or biologically active agents. FIG. 1 is an example ofa schematic of one embodiment of the device, but the device is notintended to be bound to just this design. Rather, the design can includevariations such as those routine in the art. In some embodiments, devicedesign can be modified depending on the type of biologically activeagents and/or cells encapsulated and to meet the needs and function ofthe study. A device of any size or shape reasonable can be furthercompartmentalized into having at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, a least 14, at least15, at least 16, least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24 or more chambers orcompartments. One purpose for creating a plurality of compartments isthat it increases the surface area for nutrient and oxygen exchangebetween the encapsulated cells and, for example and the interstitialspace surrounding the device; see FIGS. 1-11 for example. Further, suchdesigns prohibit or do not promote large cell aggregates or clusters oragglomerations such that cells packed in the center of the largeclusters/agglomerations are denied, or receive less, nutrients andoxygen and therefore potentially do not survive. Devices containing aplurality of chambers or compartments therefore are better capable todisperse the cells throughout the chamber/compartment orchambers/compartments. In this way, there is more opportunity for eachcell to receive nutrients and oxygen, thereby promoting cell survivaland not cell death.

One embodiment relates to a substantially elliptical to rectangularshape device; see FIGS. 1 and 6. These devices are furthercompartmentalized or reconfigured so that instead of a slightlyflattened device there is a weld or seam running through the center ofthe device, either sealing off each half of the device, thus forming twoseparate reservoirs, lumens, chambers, void spaces, containers orcompartments; or the weld or seam creates one U-shaped chamber which isseparated or divided in the middle due to the weld but such a weld inthis instance does not completely seal off the chambers; see FIG. 1. InFIG. 1 two ports provides for ease of filling and flushing cells intoand through the chambers.

Another embodiment relates to a similar elliptical or rectangular shapedevice having 2, 3, 4, 5, 6, 7, 8, 9, 10 or more welds across the planeof the device. In some aspects the welds are across the horizontalaspect or plane of the device. In other aspects the welds are across thevertical aspect or plane of the device. In still other aspects,intersecting welds are present across both the horizontal and verticalaspects of the plane. In some aspects the welds are parallel andequidistant to each other. In other aspects the welds are perpendicular.In still other aspects the welds are parallel but not equidistant. As inthe above example, such a design can effectively form up to 2, 3, 4, 5,6, 7, 8, 9, 10 or more chambers, wholly separated if the weld runstraverses and connects both boundaries of the device, or it can createone continuous chamber but interdigitated. Further, although certainexemplary devices are described in FIGS. 1-11 with welds being parallelor parallel and equidistant, still other devices can be customized ormade with welds in any direction or orientation, including long weldswhich have regions interrupted by no welds. The type and number of weldsused can depend on the cell population or agent employed and for whattreatment or purpose. In some embodiments, welds can be arranged tomodify the look of the device.

FIG. 1 shows an encapsulation device that embodies features describedherein, but as described above, this is just one illustration and one ofordinary skill in the art can envisage that by forming differentconfigurations using welds or seams in any such device, one cancustomize the number of compartments suitable for the purpose. FIGS. 2-5show top, side and end cross sections of the same device. The device canbe ultrasonically welded around the entire perimeter 1 to create acompletely enclosed internal lumen. Other means of sealing or wallingoff membranes to form the pouch like device can be used. The lumen isfurther compartmentalized by an internal weld 2 that is centrallylocated and extends down the long axis of the device. This weld extendsto a point 3 that effectively limits the thickness or depth of eachcompartment yet does not completely segregate the internal lumen. Bythis approach, the width and depth of the compartments are controlledand can be varied as is required to enable cell product survival andperformance. Moreover, all dimensions of the device, which include butare not limited to, the overall length, overall width, perimeter weldthickness, perimeter weld width, compartment length, compartment width,compartment depth, internal weld length, internal weld width and portposition are design specifications that can be modified to optimize thedevice for unique cell products and/or biologically active agents.

Referring to FIG. 1, the compartment is loaded with a cell product orbiologically active agent through two individual ports 5, 5′ that areincorporated into the device during ultrasonic welding of the perimeter.These ports extend into the lumen or compartments and allow access tothe compartment for the purpose of evenly distributing cells and/oragents during loading. Further, as the ports 5, 5′ are connected via theU-shaped internal lumen as in FIG. 1, gas is allowed to vent througheach port 5 while the adjacent port 5′ is being loaded, thus preventingthe accumulation of pressure in the device.

Alternatively, in another embodiment, the devices provided hereincontain no ports of entry or exit, i.e. the devices are said to beport-less. Such an embodiment is shown in FIG. 6. FIGS. 7-9 show a top,side and end cross section of a substantially similar device. A two,three or more stage welding process may be necessary to create aport-less device as that shown in FIGS. 6-11. For example, in oneaspect, the elliptical/rectangular outer perimeter 6 and thecompartmentalization spot welds 7 are first created by ultrasonicwelding. The spot welds 7 function similarly to the internal weld 2 ofFIG. 1. The spot welds 7 are placed is a manner across the device toperiodically limit the expansion of the lumen or compartment 8 at anygiven point. Again, the lumen or compartments 8 created by spot welding,therefore interconnecting the compartments 8, and not isolating orwholly separating any one lumen or compartment. Moreover, the totalnumber, diameter and distribution of the spot welds 7 are designparameters that can be optimized to accommodate the loading dynamics andgrowth rates of any cell product or agent.

Once cells are loaded into the device, the outer perimeter is completelyand aseptically sealed by a second ultrasonic weld across the edge 9 ofthe device. The result of the multi-step sealing process is thatfinished devices are totally enclosed and have no ports extending fromthe perimeter. This approach simplifies the loading process and improvesthe overall integrity and safety of the device, as the ports can be anarea of the perimeter where breaches can occur as a result of suboptimalultrasonic welding.

Further, although the above process was described in 2 sequential steps,the means for encapsulating the cells and/or agents is not limited tothe described 2 steps but to any number of steps, in any order,necessary to encapsulate the cells and at the same time prevent orreduce the level of breach of the device.

In another embodiment, FIGS. 10 and 11 show an encapsulation devicesubstantially similar to the device shown in FIG. 6, but the spot welds10 have been modified during the welding process to have the centersremoved. One of ordinary skill in the art cam accomplish this in variousways, e.g., by using an ultrasonic sonotrode that has an internalsharpened edge, which can cut the material immediately after welding.These cut-out welds 10 have an advantage in that they are more readilyintegrated with the host tissue because the cut-out welds 10 promotevascularization of the device, thus improving the survival andperformance of oxygen-dependent cell products and/or agents. As aconsequence of facilitating and promoting new vasculature through thedevice, there is improved diffusive transport of oxygen in the X-Ydirection, which is normally limited towards the center of planar sheetdevices.

In other embodiments, the device design can be different shapes, e.g.the cell encapsulation device can be in the shape of a tube or flattenedtube or any other such shape which satisfies one of the aboverequirements for a device of the invention.

Device Materials

Cell permeable and impermeable membranes comprising of have beendescribed in the art including those patents previously described aboveby Baxter or otherwise previously referred to as TheraCyte cellencapsulation devices including, U.S. Pat. Nos. 6,773,458; 6,520,997;6,156,305; 6,060,640; 5,964,804; 5,964,261; 5,882,354; 5,807,406;5,800,529; 5,782,912; 5,741,330; 5,733,336; 5,713,888; 5,653,756;5,593,440; 5,569,462; 5,549,675; 5,545,223; 5,453,278; 5,421,923;5,344,454; 5,314,471; 5,324,518; 5,219,361; 5,100,392; and 5,011,494,which are herein incorporated by reference in their entireties.

In one embodiment, the encapsulating devices are comprised of abiocompatible material including, but are not limited to, anisotropicmaterials, polysulfone (PSF), nano-fiber mats, polyimide,tetrafluoroethylene/polytetrafluoroethylene (PTFE; also known asTeflon®), ePTFE (expanded polytetrafluoroethylene), polyacrylonitrile,polyethersulfone, acrylic resin, cellulose acetate, cellulose nitrate,polyamide, as well as hydroxylpropyl methyl cellulose (PMC) membranes.These and substantially similar membrane types and components aremanufactured by at least Gore®, Phillips Scientific®, Zeus®, Pall® andDewal® to name a few.

Immobilized Device

Also provided is an implantable device, which is immobilized at animplantation site to maintain the encapsulated cell and/or biologicalactive agent at the implantation site and permit diffusion of, forexample, an expressed and secreted therapeutic polypeptide from theimplantation site. In one aspect, the implantation site is at, or closein proximity to, the tissue or organ which is focus of the treatment. Inother aspects, where delivery of the secreted agent from the device isnot location dependent and biodistribution of the agent is dependent onthe vasculature, the device can be implanted in a remote location. Forexample, in a preferred embodiment, the biocompatible device isimplanted subcutaneously under the skin on the forearm, or flank, orback, or buttocks, or leg and the like, where it substantially remainsuntil such time as it is required for it to be removed.

Expandable Devices

Devices described herein have inner and outer surfaces wherein thedevice contains at least one void (or reservoir, or lumen, or containeror compartment) and wherein at least one void is open to the innersurface of the device. Conventional implantable devices are commonlymade of rigid, non-expandable biocompatible materials. One embodiment ofthe device described herein is made of an expandable material. Otherembodiments are directed to non-expandable materials. Whether the deviceis capable of expanding may be an inherent part of the materialsemployed to make the device, e.g., a polymer sheath which is expandable,or can be designed such that they are expandable or have expandablecapabilities. For example, a device which expands in size to houseadditional cells or to refill an existing device is provided.

In another embodiment, the implantable device is contained in a housingor holder, which is slightly more rigid, and non-expandable but allowingsufficient means to increase cell or agent capacity by increasing thenumber of or implant devices. For example, means for inserting anadditional reservoir, lumen, container, compartment or cassette eachhaving pre-loaded cells or agent. Alternatively, the housing contains aplurality of devices only some of which are loaded with cells or havecells encapsulated therein, while others are empty, which can be loadedand filled with cells or agents at a later period in time or any timesubsequent the initial implantation. Such an expandable housing iscomprised of inert materials suitable for implantation in the body, e.g. metal, titanium, titanium alloy or a stainless steel alloy, plastic,and ceramic appropriate for implantation in the mammal, morespecifically, the human body.

Still in another embodiment, such a housing or implant device holderincludes an outer sleeve having a longitudinal axis, at least onepassage along the longitudinal axis, and a distal end and a deviceengagement area adapted to cooperatively engage the device. As ananalogy, the device holder functions similarly to a disk or cassetteholder capable of housing more than one disk or cassette at any one timeor for a long period of time. In still another embodiment, the deviceholder contains an expander adapted to increase the height of the holder

Refillable Cell Encapsulation Devices

Another embodiment relates to an encapsulation device with a refillablereservoir, lumen, container or compartment, which can be periodicallyfilled or flushed with appropriate therapeutic or biologically activeagents and/or cells. Such filling may be accomplished by injecting atherapeutically effective amount of the appropriate therapeutic orbiologically active agents and/or cells into an implanted reservoir,lumen, container or compartment, e.g., subdermally or subcutaneouslyusing a syringe or other standard means in the art for filling likereservoirs, lumens, containers or compartments in vivo.

Encapsulated Cells

In some embodiments, the system comprises a cell density between about1×10⁵, 1×10⁶ cells/ml to about 1×10¹⁰ cells/mL or more. In someembodiments, the cell survives under culture conditions or in vivo inthe system for at least a month, two months, three months, four months,five months, six months, seven months, eight months, nine months, tenmonths, eleven months, twelve months or a year or more with afunctionality that represents at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of the function expressed at thetime the cells are/were introduced into the system or at the time thecells fully develop and/or mature in the system, e.g. implantation ofprogenitor cells which need to further develop or mature to functionalcells in vivo. In some embodiments, the cell in the system expands insaid system to increase in cell density and/or cell function uponimplantation of the system in vivo.

Methods for Increasing Cell Viability

One obstacle to the field of cell and tissueencapsulation/immuno-isolation has been the lack of sufficient oxygenand nutrient transport across the polymer membranes used to encapsulatecells and tissues. The result of this insufficient gas and nutrientexchange is lowered metabolic activity and cell death. Embodimentsdescribed herein relate to an implantable cell encapsulation deviceaddressing this drawback of the prior art.

Oxygen partial pressures have been measured within islets, in theirnative environment, after isolation, and post-transplant in variouspolymer devices as well as naked or free, for example, under the kidneycapsule. Oxygen partial pressures in pancreatic islets are the highestof any organ in the body (37-46 mmHg). However, upon isolation, thesevalues fall drastically (14-19 mm Hg). Upon transplantation ofpancreatic islets into normo-glycemic animals the values decreaseslightly (9-15 mmHg) as compare to their isolated values. See Dionne etal., Trans. Am. Soc. Artf. Intern. Organs. 1989; 35: 739-741; andCarlsson et al., Diabetes July 1998 47(7):1027-32, the disclosure ofwhich is herein expressly incorporated by reference. These studiesdemonstrate that when tissues are immuno-isolated and transplanted, evenin a vascularized region such as the kidney capsule, the oxygen partialpressures drop as compared to their native states (37-46 mmHg). Hence,these nearly anoxic conditions can result in cell death, particularlythe nearer the cell to the core of a cell cluster or core of anencapsulating device.

In order to achieve better oxygen availability and delivery to theencapsulated cells or tissues and/or biologically active agents,embodiments described herein relate to the use of, for example,perfluorinated substances in the device design and/or formulation, e.g.,in the membranes or materials employed for assembly of the device. Inparticular, perfluoro organic compounds, e.g., perfluorocarbons (PFCs),are good solvents because they have several fold higher solubility foroxygen than water. For example, under normal conditions, liquid PFCsdissolve between 40 and 55% by volume of oxygen and between 100 and 150%by volume of CO2. PFCs are largely used as blood substitutes and tissuepreservation. Additionally, PFC derivatives are dense, chemically inert,and water insoluble compounds that cannot be metabolized.

In another aspect of the embodiments, enhanced O₂ delivery is performedby a PFC-emulsion or mixture of PFC with some matrix. The devicecomponents or cells for example could be suspended or soaked orincubated in the emulsion/matrix to form a coating. Still certain PFCemulsions with higher weight/volume concentrations have been known tohave improved oxygen delivery and retention properties. And because ofthe higher oxygen partial pressure created by the O₂ carryingcapabilities of PFCs, an O₂ pressure gradient is created that drivesdiffusion of dissolved oxygen into the tissue, thereby enhancing O₂delivery to the cells.

The PFC substance includes but is not limited to perfluorotributylamine(FC-43), perfluorodecalin, perfluorooctyl bromide,bis-perfluorobutyl-ethene, or other suitable PFCs. Preferred PFCstypically contain about 60 to about 76 weight percent carbon-bondedfluorine. The perfluorinated fluids can be single compounds, but usuallywill be a mixture of such compounds. U.S. Pat. No. 2,500,388 (Simons);U.S. Pat. No. 2,519,983 (Simons); U.S. Pat. No. 2,594,272 (Kauck etal.); U.S. Pat. No. 2,616,927 (Kauck et al.); and U.S. Pat. No.4,788,339 (Moore et al.), the disclosures of which are hereinincorporated by reference in their entireties. PFCs useful in theembodiments described herein also include those described inEncyclopedia of Chemical Technology, Kirk-Othmer, Third Ed., Vol. 10,pages 874-81, John Wiley & Sons (1980). For example, useful PFCs includeperfluoro-4-methylmorpholine, perfluorotriethylamine,perfluoro-2-ethyltetrahydrofuran, perfluoro-2-butyltetrahydrofuran,perfluoropentane, perfluoro-2-methylpentane, perfluorohexane,perfluoro-4-isopropylmorpholine, perfluorodibutyl ether,perfluoroheptane, perfluorooctane, and mixtures thereof. Preferred inertfluorochemical liquids include perfluorohexane,perfluoro-2-butyltetrahydrofuran, perfluoroheptane, perfluorooctane, andmixtures thereof. Commercially available PFCs useful in the embodimentsdescribed herein include FLUORINERT™ fluids, e.g., FC-72, FC-75, FC-77and FC-84, described in the 1990 product bulletin #98-0211-5347-7(101.5)NPI, FLUORINERT™ fluids, (available from Minnesota Mining andManufacturing Company, St. Paul, Minn.), and mixtures thereof.

In Vivo Imaging, Capability

In one embodiment, there is provided a means for imaging or detectingthe cells inside the encapsulating devices in vivo. Imaging servesimportant roles in stem cell therapies. For example, noninvasive formsof imaging can be used to: (1) determine the presence, severity orphenotype of the cell and/or disease to be treated; (2) monitorengrafted cell therapies for the appearance of deleterious or non-targetcell types and structures, such as cysts or microcysts; (3) guide thedelivery of therapy; (4) follow the time-course of disease and evaluatethe effects or efficacy of therapy; (5) provide labels and definemechanisms of therapy; (6) analyze and evaluate survival and function ofengrafted cells; and (7) generally facilitate the process of any celltherapy, e.g. by determining the engraftment, survival, and localfunction of cell therapy, including cell therapies described herein fortreatment of diabetes by substitution and/or implanting pancreaticprogenitor cells. In addition, although cell therapies aim to decreasemorbidity/mortality, noninvasive imaging techniques as described hereinand in more detail below can serve as a useful surrogate endpoint, forexample, in preliminary trials or preclinical studies.

Any in vivo imaging technology is ideally: i) non-invasive; ii) reliablyrepetitive; iii) capable of tissue penetration up to a depth of at least3 mm; iv) resolution capabilities of no greater than 100 μm and ideallyno greater than 50 μm; v) imaging is not attentuated by devicematerials, e.g., can image through PTFE; vi) clinically compatible andnot technically cumbersome or complicated; vii) commercially available;viii) FDA approved for human use; ix) reasonably cost-effective; and x)can image cells in a reasonable period of time (e.g., seconds orminutes), or any combination of the above.

To date, current methods include but are not limited to confocalmicroscopy, 2-photon microscopy, high frequency ultrasound, opticalcoherence tomography (OCT), photoacoustic tomography (PAT), computedtomography (CT), magnetic resonance imaging (MRI), single photonemission computed tomography (SPECT) and positron emission tomography(PET). These alone or combined can provide useful means to monitor thetransplanted cells. Also, it is expected that such technologies willimprove over time but that the essential tenets of how each technologyfunctions or its utility is substantially similar. That said, in vivoimaging described herein is not intended to be limited to technologiesdescribed below but to technologies later discovered and described whichwould serve the same utility as that described herein.

In one embodiment, the imaging technique employed would be non-invasiveand provide for a 3-dimensional tomographic data, have high temporal andspatial resolution, allow molecular imaging, and would be inexpensiveand portable. While at present no single modality is ideal (discussed inmore detail below), each has different attributes and these modalitiestogether can provide complimentary information.

Confocal microscopy is an optical imaging technique that increasesmicrograph contrast and is capable of reconstructing three-dimensionalimages by using a spatial pinhole to eliminate out-of-focus light inspecimens that are thicker than the focal plane. Since only one point inthe sample is illuminated at a time, 2D or 3D imaging requires scanningover a regular raster (i.e. a rectangular pattern of parallel scanninglines) in the specimen. Three principal scanning variations are commonlyemployed to produce confocal microscope images. Fundamentally equivalentconfocal operation can be achieved by employing a laterally translatingspecimen stage coupled to a stationary illuminating light beam (stagescanning), a scanned light beam with a stationary stage (beam scanning),or by maintaining both the stage and light source stationary whilescanning the specimen with an array of light points transmitted throughapertures in a spinning Nipkow or Nipkov disk. Each technique hasperformance features that make it advantageous for specific confocalapplications, but that limits the usefulness of that feature for otherapplications.

All confocal microscopes rely on the ability of the technique to producehigh-resolution images, termed optical sections, in sequence throughrelatively thick sections or whole-mount specimens. Based on the opticalsection as the basic image unit, data can be collected from fixed andstained specimens in single, double, triple, or multiple-wavelengthillumination modes, and the images collected with the variousillumination and labeling strategies will be in register with each otherLive cell imaging and time-lapse sequences are possible, and digitalimage processing methods applied to sequences of images allow z-seriesand three-dimensional representation of specimens, as well as thetime-sequence presentation of 3D data as four-dimensional imaging. Theuse of above confocal microscopes is not limiting as other confocalmicroscopes now or later discovered are also encompassed in theembodiments described herein.

A large number of fluorescent probes are available that, whenincorporated in relatively simple protocols, can stain certain cellularsurface markers and/or proteins and intracellular organelles andstructures, e.g., Celltracker, DiI, nuclear vital dyes, and the like.Fluorescent markers which specifically bind directly or indirectly tocertain cell surface markers can be especially useful for identificationof for example unwanted cell types. In one preferred embodiment, realtime in vivo imaging for the presence of encapsulated pluripotent cellsprovides a means to detect, and therefore the potential to prevent,teratoma formation caused from pluripotent stem cells, such as hES orhuman embryonic gonadal cells or induced pluripotent stem (IPS) cells orparthenote cells and the like. The same means of detection can alsoidentify pluripotent Stem cells which have escaped or leaked out of thedevice (or become un-encapsulated). Identification of such cells canalso be performed using fluorescently labeled promoter genes OCT4 andNANOG that are up-regulated in expression in pluripotent stem cells.Similarly, certain intracellular fluorescent markers that label nuclei,the Golgi apparatus, the endoplasmic reticulum, and mitochondria, andeven dyes such as fluorescently labeled phalloidins that targetpolymerized actin in cells, are also commercially available and canprovide critical information about the fate of a cell.

In another embodiment, two-photon excited fluorescence (TPEF) microscopyis a noninvasive means to monitor differentiation or, stated in thereverse, to identify pluripotent stem cells (e.g., hESCs or IPS cells orparthenote cells) which did not differentiate and were inadvertentlyimplanted as a very small percentage of the product cells that wereencapsulated in the device described herein. Two-photon excitedfluorescence microscopy relies substantially on endogenous sources ofcontrast, but can also detect, for example, fibrillar matrix moleculesvia second harmonic generation. In brief, two-photon microscopy relieson fluorescence emission similar to that employed by confocalmicroscopy. Rice et al. (2007) described that TPEF can be used to revealquantitative differences in the biochemical status and the shape ofdifferentiating and nondifferentiating stem cells in two-dimensional(2-D). See Rice et al. (2007) J Biomed Opt. 2007 November-December;12(6), the disclosure of which is expressly incorporated by referenceherein. In one embodiment, pluripotent stem cells can be geneticallymodified to express a fluorescent protein, e.g., enhanced greenfluorescence protein, and driven by a pluripotent stem cell promoter(e.g., OCT4 or NANOG or any other pluripotent stem cell promoter lateridentified). For those implantable devices that are deeper thansubcutaneous implants, i.e. deep below the skin surface, two-photonprovides for a non-invasive deeper imaging than confocal microscopy.Further, the infrared light used is less harmful to living cells thanvisible or ultraviolet exposure, as the photon energy required forfluorescence excitation only occurs at the plane of focus and is notexperienced by cells or tissues in the out-of-focus planes.

In still another embodiment, ultrasound is portable, essentiallyharmless, versatile, and can be done in real-time at the time ofimplantation of the encapsulated cell product and/or encapsulatedbiologically active agent. In particular, high frequency ultrasound suchas that described by VisualSonics. High-resolution imaging enables invivo assessment of anatomical structures and hemodynamic function inlongitudinal studies of mammal. For example, Vevo by VisualSonicsoffers: (1) ability to perform longitudinal studies of diseaseprogression and regression in individual subjects; (2) image resolutionof anatomical and physiological structures of down to 30 microns; (3)ability to visualize image-guided needle injection and extraction; (4)microcirculatory and cardiovascular blood flow assessment; (5) highthroughput via user-friendly equipment and research-driven interface;and (6) open architecture allowing comprehensive measurement andannotations and offline data analysis. The ability to assessmicrocirculatory and cardiovascular blood flow will assist indetermining the viability of the cells, e.g. O₂ flow and delivery.

In another embodiment, magnetic resonance imaging (MRI) can be utilizedto distinguish between healthy and diseased tissue using a contrastagent. Yet, in another embodiment, computerized tomography (CT) or CTscans can be used to create a detailed picture of the body's tissues andstructure. Again here, a contrast agent is utilized and makes it easy tovisualize abnormal tissue due to specific absorption rates. One use of acontrast agent such as Indium-111 (I-111) oxine is for tracking stemcells although it does have a short half-life. Still, in anotherembodiment, Positron Emission Tomography (PET) scans can be used tomeasure emissions from positron-emitting molecules e.g., carbon,nitrogen, and oxygen to name a few, and provide valuable functionalinformation. In yet another embodiment, optical coherence tomography(OCT) or photoacoustic tomography (PAT) may also be used to examinecells and tissues inside and outside the device. OCT detects differencesin the reflectivity of various tissues while PAT detects ultrasonicwaves created when tissues are heated by exposure to low energy laserlight.

Various methods and techniques or tools, alone or combined, can beemployed to visualize, analyze and assess the implanted cells inside thedevice in vivo. These and other technologies now known or laterdeveloped can be utilized to the extent they allow for in vivo imagingand monitoring of the cells and/or agent as described herein.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in their entirety in order to more fullydescribe the state of the art to which this patent pertains.

Example 1 Encapsulated Pancreatic Progenitors Function In Vivo

The following example was performed, at least in part, to firstdetermine the integrity of methods of encapsulating pancreaticprogenitor cells, including a bio-compatible device and amedical/mechanical device; and second to determine whether whollyencapsulated pancreatic progenitor cells survive and mature tofunctioning hormone-secreting cells in vivo as compared tounencapsulated pancreatic progenitor cells (controls).

Methods for producing pancreatic cell lineages from human embryonic stem(hES) cells are substantially as described in U.S. Pat. No. 7,534,608,entitled METHODS OF PRODUCING PANCREATIC HORMONES, U.S. application Ser.No. 12/264,760, entitled STEM CELL AGGREGATE SUSPENSION COMPOSITIONS ANDMETHODS OF DIFFERENTIATION THEREOF, filed Oct. 4, 2008; U.S. applicationSer. No. 11/773,944, entitled METHODS OF PRODUCING PANCREATIC HORMONES,filed Jul. 5, 2007; U.S. application Ser. No. 12/132,437, GROWTH FACTORSFOR PRODUCTION OF DEFINITIVE ENDODERM, filed Jun. 3, 2008; U.S.application Ser. No. 12/107,020, entitled METHODS FOR PURIFYING ENDODERMAND PANCREATIC ENDODERM CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS,filed Apr. 8, 2008; U.S. application Ser. No. 11/875,057, entitledMETHODS AND COMPOSITIONS FOR FEEDER-FREE PLURIPOTENT STEM CELL MEDIACONTAINING HUMAN SERUM, filed Oct. 19, 2007; U.S. application Ser. No.11/678,487, entitled COMPOSITIONS AND METHODS FOR CULTURING DIFFERENTIALCELLS, filed Feb. 23, 2007; U.S. Pat. No. 7,432,104, entitledALTERNATIVE COMPOSITIONS & METHODS FOR THE CULTURE OF STEM CELLS; Kroonet al. (2008) Nature Biotechnology 26(4): 443-452; d'Amour et al. 2005Nat Biotechnol. 23:1534-41; D'Amour et al. 2006 Nat Biotechnol.24(11):1392-401; McLean et al., 2007 Stem Cells 25:29-38, which are allherein incorporated in their entireties by reference.

Briefly, undifferentiated human embryonic stem (hES) cells weremaintained on mouse embryo fibroblasts feeder layers (Specialty Media)in DMEM/F12 (Mediatech) supplemented with 20% KnockOut serum replacement(KOSR, GIBCO BRL), 1 mM nonessential amino acids (GIBCO BRL), Glutamax(GIBCO BRL), penicillin/streptomycin (GIBCO BRL), 0.55 mM of2-mercaptoethanol (GIBCO BRL) and 4 ng/mL recombinant human FGF2 (R&DSystems) and alternatively supplemented in 10-20 ng/mL of Activin A (R&DSystems). Human ES cell cultures were manually passaged at about 1:4 to1:8, 1:9, or 1:10 split ratio every 5 to 7 days. Prior todifferentiation either as adherent cultures or in cell aggregatesuspensions, they were given a brief wash in PBS^(+/+) (containing Mg⁺⁺and Ca⁺⁺, Invitrogen). Human ES cell lines can include, but are notlimited to, CyT49, CyT203, Cyt25, BG01 and BG02.

Methods for culturing and differentiating cells or cell populations insuspension are described in detail in International ApplicationPCT/US2007/062755, COMPOSITIONS AND METHODS FOR CULTURING DIFFERENTIALCELLS, filed 23 Feb. 2007 and U.S. application Ser. No. 12/264,760, STEMCELL AGGREGATE SUSPENSION COMPOSITIONS AND METHODS OF DIFFERENTIATIONTHEREOF, filed 4 Nov. 2008, which are herein incorporated by referencein their entireties.

The differentiation culture conditions were substantially similar tothat described in D'Amour et al. 2006, supra, and Example 4 below, bothdescribing a 5 step differentiation protocol: stage 1 (definitiveendoderm; d 1-d 4), stage 2 (primitive gut tube or foregut endoderm; d 5to d 8), stage 3 (posterior foregut or Pdx1-positive endoderm; d 9 to d12), stage 4 (pancreatic progenitor, pancreatic epithelium and/orendocrine precursor; d 13 to d 15) and stage 5 (hormone expressingendocrine cell, d 16 or more).

At stage 4, retinoic acid (RA) was withdrawn from the stage 3 cultures,the cultures were washed once with DMEM plus B27 (1:100 Gibco), and thenthe wash was replaced with either DMEM+1XB27 supplement alone or withany combinations of or any or all of the following factors: Noggin (50ng/ml), FGF10 (50 ng/ml), KGF (25-50 ng/ml), EGF (25-50 ng/ml), 1-5% FBSfor 4-8 days. In cases where no RA was added, noggin at 30-100 ng/mL(R&D systems) was added to the media for 1-9 days. Alternatively, noadditional growth factors were added at stage 4. Also, cell-survivalagents such as Y-27632, fasudil, H-1152P, and a mixture comprisinginsulin/transferrin/selenium (ITS) can be added to the cultures.

Regardless of whether the pancreatic progenitors were produced fromadherent cultures or in cell aggregate suspensions, all pancreaticprogenitor cell populations when transplanted in mammals developed andmatured into functional endocrine tissues in vivo. In vivo production ofinsulin by the hES-derived transplanted cells is described in the U.S.Applications and references above, e.g., U.S. application Ser. No.11/773,944, entitled METHODS OF PRODUCING PANCREATIC HORMONES and Kroonet al. 2008, supra.

Unlike the cell compositions described in U.S. application Ser. No.11/773,944 and Kroon et al. 2008 supra, the pancreatic progenitors inthis study were wholly isolated or encapsulated in vivo. Pancreaticprogenitor cells were encapsulated using a bio-compatible polyethyleneglycol (PEG), which is described in more detail in U.S. Pat. No.7,427,415, entitled IMPLANTATION OF ENCAPSULATED BIOLOGICAL MATERIALSFOR TREATING DISEASES, which is herein incorporated by reference.PEG-encapsulated pancreatic progenitors were transplanted under theepididymal fat pad (EFP); serum C-peptide levels at various time pointspost glucose-stimulation were determined; and immunohistochemicalanalysis was done on the PEG-encapsulated explants. Again, these methodshave been previously described in U.S. application Ser. No. 11/773,944,entitled METHODS OF PRODUCING PANCREATIC HORMONES and Kroon et al. 2008,supra. (data not shown). Immunohistochemical analysis showed that thepancreatic progenitor cells were capable of maturing in vivo andcontained hormone expressing cells such as insulin, glucagon andsomatostatin.

Encapsulation of the pancreatic progenitor cells was also performedusing a medical or mechanical device, e.g., a TheraCyte cellencapsulation device. All references to TheraCyte cell encapsulationdevices are to devices that were purchased directly from themanufacturer (Theracyte, Inc., Irvine, Calif.) and are further describedin U.S. Pat. Nos. 6,773,458; 6,156,305; 6,060,640; 5,964,804; 5,964,261;5,882,354; 5,807,406; 5,800,529; 5,782,912; 5,741,330; 5,733,336;5,713,888; 5,653,756; 5,593,440; 5,569,462; 5,549,675; 5,545,223;5,453,278; 5,421,923; 5,344,454; 5,314,471; 5,324,518; 5,219,361;5,100,392; and 5,011,494, which are all herein incorporated in theirentireties by reference. Pancreatic progenitor cells were either loadedinto the devices ex vivo, or once the devices had been implanted for aperiod of time to allow for prevascularization of the device, then thecells were loaded in vivo via the loading port on one side of thedevice.

Therefore, the device contains a first membrane which is impermeable tocells (0.4 microns) but at the same does not restrict movement of oxygenand various nutrients in and out of the inner membrane, e.g. glucosefrom outside the inner membrane can permeate into the capsule containingthe mature pancreatic hormone secreting cells, which in response to theglucose, can secrete insulin which then permeates out of the innermembrane. The device also contains an outer vascularizing membrane.

In order to use a device for any cell therapy, the device has to whollycontain the cells in vivo (e.g., immuno-isolate the hES-derived cellsfrom the host). To determine the integrity of the TheraCyte device,intact devices containing the pancreatic progenitor cells were comparedto those devices which had perforated holes in the membranes in vivo.Perforating holes into the devices allows for host cellular invasion andtherefore establish host-graft cell-to-cell contact.

Two 4.5 μL TheraCyte devices were first prevascularized by surgicallyimplanting them under the epididymal fat pads (EFP) or subcutaneously(SQ) in each male severe combined immunodeficient (SCID)-beige (Bg)mice. That is, one animal received 2 devices under the EFP, and anotheranimal received 2 devices SQ These intact but empty (no pancreaticprogenitor cells) devices remained in the animal for a sufficient periodof time allowing for host vasculature structures to form and associatewith the device, e.g., at least 2 to 8 weeks. After 8 weeks, about1.5×10⁶ cells pancreatic progenitor cells derived from hES cells wereloaded into each of the 4 devices. At the same time as the animals withthe prevascularized devices were being loaded, 3 other animals wereimplanted with two modified Theracyte devices wherein an originalTheracyte device of the same size (4.5 μL) was modified withperforations in the membranes of the device. These perforated devices (2perforated devices per animal) were loaded with cells ex vivo with aboutthe same dosage of cells as was loaded into of the perforated devices.Also at the same time, two positive controls were carried along sidethese experiments and both animals were grafted with pancreaticprogenitors on Gelfoam as described in U.S. application Ser. No.11/773,944, entitled METHODS OF PRODUCING PANCREATIC HORMONES and Kroonet al. 2008, supra, although in one animal two grafts were placed underthe EFP and in the other animal only one graft was placed in the EFP.Table 1 summarizes the results of the above experiments.

TABLE 1 Human C-peptide serum levels from encapsulated mature pancreatichormone-secreting cells 9 week 12 week 15 week GSIS Human Human HumanTime C-pep GSIS C-pep GSIS C-pep Implant Animal # (min) pM Time pM TimepM PV 675 0 91 0 186 EFP 60 224 30 304 2 × 1.5M 60 419 PV 676 0 157 0322 0 908 SQ 60 610 30 532 5 874 2 × 1.5M 60 2637 30 3037 nPV + 679 0239 0 374 holes 60 727 30 482 EFP 60 2259 2 × 1.5M 680 0 218 0 329 60899 30 506 60 2177 681 0 408 0 422 0 1554 60 2136 30 1059 5 1615 60 275130 10330 EFP GF 682 0 912 0 488 0 1504 2 × 1.5M 60 3716 30 3025 5 187860 3673 30 4288 EFP GF 684 0 279 0 444 0 1498 1 × 1.5M 60 580 30 751 51411 60 3000 30 4698 With respect to glucose stimulated insulinsecretion (GSIS), 0 refers to time 0; 5 refers to 5 minutes post glucosestimulation; 30 refers to 30 minutes post glucose stimulation; 60 refersto 60 minutes post glucose stimulation; PV TC EFP, prevascularizedTheraCyte under the epididymal fat pad; PV TC SQ, prevascularizedTheraCyte subcutaneous; nPV TC + holes EFP, non-prevascularizedTheraCyte perforated under the epididymal fat pad; and EFP GF,epididymal fat pad on Gelfoam, 2 × 1.5M, two constructs withapproximately 1.5 × 10⁶ cells.

The pancreatic progenitor cells were allowed to develop and mature invivo and insulin secretion and glucose responsiveness of the now maturehormone-secreting cells were determined substantially as described inU.S. application Ser. No. 11/773,944, entitled METHODS OF PRODUCINGPANCREATIC HORMONES and Kroon et al. 2008, supra. See Table 1.Additionally, to determine the integrity of the devices, some animalswere sacrificed and immunohistochemical examination of the devices wasperformed.

Applicants previously demonstrated that serum human C-peptide levelsbelow 50 pM, or insulin levels below 25 pM, are insignificant todemonstrate that insulin-secreting cells are responsive to glucose invivo. This same standard was used in these studies. The results of thestudies are shown in Table 1. Both the original Theracyte device and themodified Theracyte device after 8, 12 and 15 weeks had comparable serumhuman C-peptide levels (animal nos. 675-676 & 679-681), with theexception of animal number 681 at 30 minutes post glucose stimulationwhereby the serum C-peptide levels was much higher than any other animalat that time period.

First with regard to the integrity of the TheraCyte encapsulatingdevice, standard hematoxylin and eosin stains of the original Theracytedevice and the modified Theracyte device (animal nos. 675-676 and679-681, respectively) were performed. Microscopic examination of thesedevices showed that the original Theracyte devices have various hostvasculature structures including vascular type cells surrounding thedevice, but these similar structures were not observed invading theinner cell impermeable membrane and into the space containing thehES-derived cells. That is, there was no host vasculature structuresobserved inside the inner cell impermeable membrane housing thehES-derived cells, or the graft. In contrast, microscopic examination ofthe modified Theracyte devices showed that not only was there hostvasculature structures associated on the outside of the device, butthere were vasculature structures and vascular cells found inside theperforated inner cell impermeable membrane. Hence, the originalTheraCyte devices can wholly contain the hES-derived cells and hostcells and tissues were not observed in the space housing the hES-derivedcells.

In summary, the TheraCyte device is capable of wholly encapsulating(isolating) the hES-derived cells in vivo and the pancreatic progenitorscan survive and mature to functioning hormone-secreting cells in vivo inthese devices.

In addition to demonstrating the integrity of the TheraCyte device, thepresent studies also demonstrate that the wholly intact devices allowfor sufficient oxygen and various nutrients exchanged between thecontained hES-derived cells and the host milieu, and the pancreaticprogenitors are capable of surviving and maturing in vivo. For example,serum human C-peptide levels in the prevascularized devices at 9 and 12weeks were not as robust as the equivalent time point as compared to thecontrols (animals 682 and 684). However, by the 15^(th) week(post-implant with cells), serum human C-peptide levels in theprevascularized devices were comparable to the unencapsulated (Gelfoam)controls.

Further, animals with the original Theracyte (prevascularized) deviceswere sacrificed and the devices (or explants) extracted (animal nos. 675& 676). Immunohistochemistry was performed substantially again asdescribed in and Kroon et al. 2008, supra by fixing the extracteddevices and/or the explants and cutting the 10-sections into thinmicrometer sections. Sections were washed with PBS twice, followed byPBST (PBS/0.2% (wt/vol) Tween20; Thermo Fisher Scientific). Blocking wasdone for 1 h at 24° C. with 5% normal donkey serum (Jackson ImmunoResearch Labs)/PBSTr (PBS/0.1% (wt/vol) Triton X-100 (Sigma)). Primaryand secondary antibodies were diluted in 1% BSA (Sigma)/PBSTr forgrafts. Primary antibodies were incubated at 4° C. overnight andsecondary antibodies for about 1 h 15 min in a moisture chamber. Thefollowing primary antibodies and dilutions were used; guinea piganti-insulin (INS), 1:500 (Dako, A0564); rabbit anti-somatostatin (SST),1:500 (Dako, A0566); goat anti-somatostatin (SST), 1:300 (Santa CruzBiotechnology, SC-7819); goat anti-glucagon (GCG), 1:100 (Santa CruzBiotechnology, SC-7780). Imaging was done by confocal microscopy (Nikon,Eclipse 80i, Ci).

Immunohistochemical examination of the original Theracytedevices/explants clearly demonstrated singly-positive hormonal cells,e.g., GCG, INS and SST expressing cells. This data supports the serumhuman C-peptide data demonstrating glucose responsiveness of thetransplanted hES-derived cells. The presence of hormone-secreting cellsdemonstrates that pancreatic progenitors are capable of survival andmaturation in vivo, even when wholly encapsulated.

The above studies clearly demonstrate the efficacy of both the originaland modified TheraCyte devices to wholly contain the hES-derivedpancreatic progenitor cells without host cellular invasion across theinner cell impermeable membrane. These studies also demonstrate that thedevices inner cell impermeable membrane, although impermeable to cells,is permeable to oxygen and various nutrients required for hES-derivedpancreatic progenitor survival in the device such that the progenitorcells are capable of maturing to hormone-secreting cells in vivo, whichare cells are function and are responsive to glucose.

Further, it is envisioned that the pancreatic-lineage cell populations,in particular, at least the pancreatic progenitors described herein,will also mature and function in vivo when encapsulated in the improveddevices, for example at least those described in FIGS. 1-11.

Example 2 Encapsulated Pancreatic Progenitors Function In Vivo in the inthe Absence of Host-Graft Cell Contact

To determine whether host-graft cell-to-cell contact was required for invivo functioning of transplanted pancreatic progenitor cell populations,cells were loaded into non-prevascularized cell encapsulation devices.

Pancreatic progenitor cell populations were generated substantially asdescribed above in Example 1. No devices in this study wereprevascularized, and all TheraCyte devices (4.5 μL) were loaded ex vivowith at least 1.5×10⁶ cells (1.5M) or 4.5×10⁶ cells (4.5M) in eachdevice. Three devices containing 1.5M cells were implantedsubcutaneously (TC SQ 1.5M), and 3 devices containing 4.5×10⁶ cells(4.5M), or about 15 μL, were implanted subcutaneously (TC SQ 4.5M) exvivo. In contrast and as controls, animals with implanted unencapsulatedpancreatic progenitors were carried along side the encapsulated, but notprevascularized, experiments. Three mice were each implantedsubcutaneously with two Gelfoam constructs loaded with about 1.9-2.4×10⁶cells (total for two constructs), or about 4 μL/construct, and 2 micewere implanted under the EFP with two Gelfoam constructs loaded withabout with about 1.9-2.4×10⁶ cells (total for two constructs), or about4 μL/construct. Table 2 summarizes the results of the above experiments.

TABLE 2 Human C-peptide serum levels from encapsulatednon-prevascularized mature pancreatic hormone-secreting cells GSIS 6 wk8.5 wk 10 wk time Cpep Cpep Cpep Implant Animal # (min) (pM) (pM) (pM)SQ 1.5M 833 0 nd 3.5 20.9 60 5.8 60.9 167.7 834 0 nd 0.3 32.5 60 0.632.6 97.5 835 0 102.8 168.3 153.1 60 77.2 197.8 440.0 SQ 4.5M 836 0 nd11.4 39.3 60 nd nd 29.9 837 0 6.8 60.9 85.8 60 8.3 137.3 188.4 838 0 4.221.6 64.4 60 39.5 36.4 98.0 SQ GF 819 0 nd 26.9 0.4 1.9-2.4M 60 4.2 26.979.4 820 0 nd 4.7 11.8 60 4.2 20.7 12.9 821 0 nd 10.9 12.3 60 2.7 69.154.3 EFP GF 822 0 nd 22.5 57.3 1.9-2.4M 60 4.7 95.0 132.8 823 0 nd 11.833.5 60 46.0 243.3 170.3 With respect to glucose stimulated insulinsecretion (GSIS), 0 refers to time 0; 60 refers to 60 minutes postglucose stimulation; SQ, subcutaneous device; SQ GF, subcutaneousGelfoam, EFP GF, EFP, epididymal fat pad Gelfoam; 1.5M, 1.5 × 106 cells;4.5, 4.5 × 106 cells; 1.9-2.4M, 1.9-2.4 × 106 cells; nd, none detected.

Although Example 1 demonstrated that hES-derived cells can survive,mature and function in vivo in prevascularized devices, based on Table 2prevascularization is not essential for cell survival, growth and/ormaturation. Table 2 compares encapsulated pancreatic progenitor cellswith unencapsulated pancreatic progenitor cells on Gelfoam, the laterhas been well documented to produce functioning hormone secreting cellsin vivo, see Kroon et al. 2008 supra. In fact, at 60 minutes postglucose-stimulation, serum C-peptide levels from the encapsulated cellswere comparable to serum C-peptides levels observed from cells whichwere unencapsulated. Compare animal numbers 833-838 to animal numbers819-823. In fact, the encapsulated cells performed better than theunencapsulated when implanted subcutaneously, e.g. compare animalnumbers 833-835 (TC SQ 1.5M) to 819-821 (SQ 1.9-2.4M). Thus, host-graftcell-to-cell contact is not essential because as clearly demonstrated inthis example, transplanted wholly encapsulated cells survive, grow andmature in the absence of any host-graft cell-to-cell contact alltogether.

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the patent. Changes,alternatives, modifications and variations therein and other uses willoccur to those skilled in the art which are encompassed within thespirit of the invention and are defined by the scope of the disclosure.For example, TheraCyte devices come in 4.5 μL, 20 μL, and 40 μL sizes,and therefore one of ordinary skill in art can scale-up the abovestudies if employing a device which is capable of containing more cells.Further, since Kroon et al. 2008 supra has demonstrated the efficacy ofpancreatic progenitors in rescuing streptozotocin (STZ) induced diabeticmice before and after graft implantation, one of ordinary skill in theart can perform analogous studies using the encapsulated cells describedherein. Also, methods of purifying or enriching for certain hES-derivedpopulations are described in detail in U.S. application Ser. No.12/107,020, entitled METHODS FOR PURIFYING ENDODERM AND PANCREATICENDODERM CELLS DERIVED FROM HES CELLS, filed Apr. 8, 2008, which isherein incorporated by reference in its entirety. Thus, one of ordinaryskill in the art can enrich for specific hES-derived cells including butnot limited to, pancreatic progenitor cells, pancreatic endocrineprecursor cells and/or endocrine precursor cells.

Example 3 Cryopreserved Pancreatic Progenitors when Implanted Developand Function In Vivo

Because cell transplantation is hindered by the lack of available cellsources and operational and logistical problems, there is a need toprovide an unlimited cell source for transplantation at times convenientto the patient.

Human ES cells were differentiated substantially as described inExamples 1 and 2 and Kroon et al., 2008, supra, as well as in Tables3α-h below. At day 14 of differentiation, the pancreatic progenitorswere centrifuged and then resuspended in freezing media containing DMEMwith 30% Xeno-free Knockout Serum Replacement, 25 mM HEPES and 10%dimethyl sulfoxide solution. Cells were aliquoted into freezing vials.Cells were equilibrated in freezing medium for about 15 minutes atambient temperature, then 45 minutes at 4° C., then placed on ice andput in a programmed freezer which was equilibrated to 0° C.

The cells and the freezing chamber were brought to −9° C. at a rate of2° C./min. The chamber and the sample were held at this temperature forabout 10 minutes, and the vials were seeded manually. The sample washeld at −9° C. for about 10 minutes and then cooled at a rate of 0.2°C./minute until the sample reached −40° C. The freezing chamber wassubsequently cooled at a rate of 25° C./minute until the sample reachedabout −150° C. The vialed cells were then moved to the vapor phase of aliquid nitrogen storage freezer.

At desired times, the vials was rapidly thawed by transferring the cellsto a 37° C. water bath. The cells were transferred to a 15 ml steriletube, containing DMEM with B-27 (1:100) and KGF+EGF (each at 50 ng/mL),mixed gently and spun briefly at 50×g. Supernatant was removed and cellswere resuspended in the same buffer plus DNAse at 25 μg/mL and placed inrotation culture.

Cell survival was quantitated by photographing the pancreatic progenitoraggregates when they have been swirled to the center of the tissueculture well, promptly upon thawing before any significant cell loss hasoccurred, and at 4 days post-thaw when the decrease in cell mass hascompleted. The area occupied by the cells in the photographs wasquantitated, and expressed as a percent survival at 4 days post-thaw. Inthis example, at least 52% survival was obtained. The morphology ofcultured pancreatic progenitor cells after cryopreservation and thawingwas identical to that of fresh cells.

After 4 days of post-thaw culturing, the cells were loaded in devicessubstantially as described above and surgically implanted in the mammalas described above. The cryopreserved cells were capable of developingand maturing into functioning hormone secreting and acinar cells of thepancreas in vivo similar to that described for fresh pancreaticprogenitor cell aggregates. See Example 4.

Hence, cryopreservation of in vitro human pancreatic progenitors derivedfrom hES cells has little or no effect on development afterimplantation. Thus, cryopreservation proves to be a reliable method ofstoring hESC-derived pancreatic progenitor cells suitable fortransplantation.

Example 4 Methods of Providing for Human Pancreatic Progenitors for theTreatment of Diabetes Pluripotent Stem Cell Culture Conditions

Culturing, proliferation and maintenance of pluripotent stem cells, inparticular ES and IPS cells, are performed substantially as described inD'Amour et al. 2005 & 2006 and Kroon et al. 2008, supra. ES base mediumof DMEM-F12/1% Glutamax/1% Non-essential amino acids/1% Pen-Strep/0.2%b-mercaptoethanol was used. For stage 0 or proliferation of hES cells,various growth factors and or insulin and insulin-like growth factorslevels were kept very low. Feeder-free pluripotent stem cells werecultured using low levels of human serum. The pluripotent stem cellswere maintained using a Rho-kinase inhibitor Y27632. It will beappreciated that other Rho-kinase inhibitors can be used with similarresults. The ES or pluripotent stem cell culture conditions aresubstantially similar to Examples 1 and 2 as described above.

It will be appreciated that the ES base medium can routinely containabout 20% Knockout Serum Replacement (KSR) or Xeno-free (XF) Knockoutserum replacement.

It will be appreciated that hES cell cultures routinely contain about 0ng/mL, about 4 ng/ml or about 10 ng/mL basic fibroblast growth factor(bFGF). As previously demonstrated, under certain conditions low levelsof Activin A help promote pluripotent stem cell proliferation withoutpromoting hES cell differentiation. Hence, pluripotent stem cellcultures typically contain about 5 ng/mL, about 10 ng/mL or about 20ng/mL of Activin A or B, or other similarly biologically active TGF-βgrowth factor families, for example, at least GDF-8 and GDF-11. Still inother pluripotent stem cell cultures, an Errb2-binding ligand such asheregulin at low levels also helps to promote hES cell proliferation,for example, at about 5 to 10 ng/mL. Also, any combination of differentor low levels of bFGF, Activin A, B or other TGF-β growth factor familymembers, specifically GDF-8 and -11, and Errb2-binding ligands such asheregulin can be employed to promote hES cell cultures, so long as lowlevels of the growth factors are maintained as to promote proliferationof hES cells and their pluripotency and not differentiation of the cellsthereof. Embodiments described herein describe various growth factors(in some cases large proteins) in maintaining and proliferatingpluripotent stem cell cultures, however, the high cost of these proteinson a large-scale manufacturing basis makes is cost-prohibitive. As such,identifying and characterizing certain small molecules to replace thelarger growth factor proteins may be beneficial. One such molecule isnor-epinephrine (NE), which is described in more detail in U.S.Application 61/172,998, titled SMALL MOLECULES SUPPORTING PLURIPOTENTCELL GROWTH AND METHODS THEREOF, and filed 27 Apr. 2009, and is hereinincorporated by reference in its entirety. In one embodiment, about 5ng/mL, about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL,about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90ng/mL, about 100 ng/mL or more is employed to maintain pluripotent stemcultures, for example, hES or iPS cultures. In one preferred embodiment,about 50 ng/mL can be employed in hES cell cultures.

It will be appreciated that proliferation of pluripotent stem cells areroutinely sustained on fibroblast feeder cells. Alternatively, ES cellscan be cultured on an extracellular matrix coated plates (Corning).Further, Bodnar et al. (Geron Corporation, Menlo Park, Calif., USA)describe growing hES cell cultures on a monolayer of extracellularmatrix, which matrix was derived by lysing fibroblast feeder layer inU.S. Pat. No. 6,800,480, the disclosure of which is herein expresslyincorporated by reference. However, in a preferred embodiment,feeder-free pluripotent stem cells are cultured using low levels ofhuman serum, for example, about 0.1%, about 0.2%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about1%, about 1.2%, about 1.4%, about 1.6%, about 1.8%, about 2% to about10% or more in a base ES cell medium. Human serum can be added to thebase media simultaneously thereby obviating any need to pre-coat tissueculture dishes as contemplated in U.S. Pat. No. 6,800,480 or thatprovided by Corning. Use of human serum for culturing, maintaining andproliferation of pluripotent stem cell cultures is described in moredetail in U.S. application Ser. No. 11/875,057, entitled METHODS ANDCOMPOSITIONS FOR FEEDER-FREE PLURIPOTENT STEM CELL MEDIA CONTAININGHUMAN SERUM, filed on 19 Oct. 2007, which is herein incorporated byreference in its entirety.

Pluripotent stem cells can also be maintained with the addition of a Rhokinase family of inhibitors, for example at least Y27632. Y27632 hasrecently been found to prevent apoptosis, as well as enhance thesurvival and cloning efficiency of dissociated human pluripotent stemcells without affecting self-renewal properties or pluripotency.Although, embodiments described herein use Y27632 due to its commercialavailability, other Rho kinase inhibitors can be employed and still bewithin the scope of the invention.

Pluripotent Stem Cell Differentiation Conditions for Stage 1

Directed differentiation of pluripotent stem cells, in particular ES andIPS cells, were performed substantially as described in D'Amour et al.2005 & 2006 and Kroon et al. 2008, supra and in above related U.S.Applications, including U.S. Application 61/171,759, titled CELLCOMPOSITIONS DERIVED FROM DEDIFFERENTIATED REPROGRAMMED CELLS, filed 22Apr. 2009, which are herein incorporated by reference in theirentireties.

Prior to differentiation Stage 1, or at day 0 of the differentiationprocess, pluripotent stem cells were cultured in a medium comprising ofRPMI1640/1% Glutamax/1% Pen-Strep and substantially no serum and/orabout 0.1% Bovine Serum Albumin (BSA). Also, 1:5000 or 1:1000 or about0.02% or 0.1%, respectively, of Insulin/Transferrin/Selenium (ITS)supplement was added. In addition, various growth factors including aTGF-β super family growth factor and a Wnt family member were added tothe differentiation medium.

It will be appreciated that the added TGF-β super family growth factorsinclude but are not limited to Activin A, Activin B, GDF-8 or GDF-11. Insome embodiments a Wnt pathway activator can be used. In anotherembodiment, Wnt-3a is used in conjunction with one of the TGF-β superfamily growth factors. In a further preferred embodiment, about 50 ng/mLof Wnt3a is employed with about 100 ng/mL of a TGF-β super family membersuch as Activin A, Activin B and GDF-8 and -11. Still in anotherembodiment, small molecules which activate similar signal transductionpathways can be substituted for the growth factors. See, for example,Borowiak, M. et al. (2009) describing two small molecules which directdifferentiation of mouse and human embryonic stem cells to endoderm.Borowiak, M. et al. (2009) Cell Stem Cell, 4(4):348-358 is hereinincorporated by reference in its entirety.

The pluripotent cells were incubated in the above media conditions forat least 24 hours, after which time the medium was exchanged to a mediumcomprising RPMI1640/1% Glutamax/1% Pen-Strep and a slight increase inFBS, approximately 0.2% FBS and further containing about 100 ng/mL of aTGF-β super family member. A Wnt family member was not added. It will beappreciated that a Wnt family member may be added to the culture afterabout 24 hours.

The cells were cultured in this medium for another 24 hours. After abouta total of 48 hours since the cells had been differentiation (day 0 today 2), the cells in the culture comprise differentiated definitiveendoderm cells.

It will be appreciated that the total number of days of differentiationin stage 1, starting with day 0 (pluripotent stem cells), can be about1-3 days, preferably about 1-2 days, and even more preferable, about 2days. It will be appreciated that after stage 1 differentiation, thecells in the culture will comprise about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, about 96%, about 97%, about 98% or about 99% differentiateddefinitive endoderm cells

Methods for determining the composition of the cultures has beenpreviously described in the above related applications, but principallyby RNA and protein assays well known in the art. Definitive endodermcells express increased levels of certain signature cell surface markerssuch as SOX17 and FOXA2, but can also express increased levels of CERand CXCR4, but do not appreciably express HNF4-α which is expressedappreciably in foregut endoderm (or PDX1-negative foregut) cells.Definitive endoderm cells also do not appreciably express markersobserved in later Stage 3, 4 or 5 cells such as or PDX1, NNF6, SOX9 andPROX 1 expressed in PDX1-positive foregut endoderm cells, or PDX1,NKX6., PTF1A, CPA and cMYC expressed in PDX1-positive pancreaticprogenitor or PDX1/NKX6.1 co-positive pancreatic progenitor cells, orNGN3, PAX4, ARX and NKX2.2 expressed in endocrine precursor cells, orINS, GCG, GHRL, SST or PP expressed in polyhormonal or singly hormonalpancreatic endocrine cells.

Differentiation Conditions for Stage 2

After about 48 hours (about 2 days) of differentiation of pluripotentstem cells to DE, the DE differentiation media was replaced by anothermedia condition which promotes human foregut endoderm (PDX1-negativeforegut endoderm) formation or Stage 2 cells. This cell culture mediumcomprises RPMI1640/1% Glutamax/1% Pen-Strep and 0.2% FBS or a furtherincrease in FBS, e.g. about 2% FBS. Similar to the above Stage 1 culturemedium, about 1:5000 or 1:1000 or about 0.02% or 0.1%, respectively, ofITS supplement was added.

It will be appreciated that the DE differentiation media is not alwayssupplemented with ITS.

However, DE differentiation growth factors such as TGF-β super familygrowth factors or Wnt family members were intentionally not included inthe medium. A TGF-β kinase inhibitor was added to the medium.

Because removal of TGF-β super family members is beneficial for properforegut endoderm formation, use of TGF-β super family member inhibitorssuch as a TGF-β kinase inhibitors, ensures that the effects of theaction of TGF-β super family members are substantially inhibited. Thisallows efficient direct differentiation of the DE to foregut endoderm(PDX1-negative foregut endoderm) without the lingering effects of DEdifferentiation in the culture.

Instead of TGF-β super family members, keratinocyte growth factor (KGF)was added to the culture to promote foregut endoderm formation. Cellswere incubated in this media for about 24 hours, after which the mediawas replaced with substantially the same media except that now the TGF-βkinase inhibitor was removed from the culture. The cells were thenincubated in this media (minus TGF-β kinase inhibitor) for 5 days withmedia changes.

It will be appreciated that the cells can be incubated in this media(minus TGF-β kinase inhibitor) for up to about 3 days for Stage 2 withpermissible media changes. The total number of days of differentiation,starting with day 0 and pluripotent stem cells, is about 3-5 days,preferably about 4-5 days, and more preferable, about 5 days.

Again, methods for determining the composition of the cultures has beenpreviously described in the above related applications, but principallyby RNA and protein assays well known in the art. Foregut endoderm cells,or PDX1-negative foregut endoderm cells, express increased levels ofcertain signature cell surface markers such as Sox17, HNF3- and HNF4-α.This is distinguished from DE of Stage 1 which does not appreciablyexpress HNF4-α, but does appreciably express the other two markers,Sox17 and HNF3-β. PDX1-negative foregut cells also do not appreciablyexpress markers observed in later Stage 3, 4 or 5 cells such as or PDX1,NNF6, SOX9 and PROX 1 expressed in PDX1-positive foregut endoderm cells,or PDX1, NKX6.1, PTF1A, CPA and cMYC expressed in PDX1-positivepancreatic progenitor or PDX1/NKX6.1 co-positive pancreatic progenitorcells, or NGN3, PAX4, ARX and NKX2.2 expressed in endocrine precursorcells, or INS, GCG, GHRL, SST or PP expressed in polyhormonal or singlyhormonal pancreatic endocrine cells.

Differentiation Conditions for Stage 3

To promote differentiation of PDX1-positive foregut endoderm cells fromPDX1-negative foregut endoderm cells of Stage 2, the PDX1-negativeforegut endoderm cell culture medium was exchanged and incubated in amedium comprising DMEM high glucose/1% Glutamax/1% Pen-Step/1% B27Supplement with either about 1 or 2 uM of Retinoic Acid (RA), about 0.25uM of KAAD-Cylcopamine and with or without about 50 ng/mL of Noggin.Alternatively, some cultures instead of receiving RA, received 1 nM toabout 3 nM of aromatic retinoid(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl]benzoicacid (TTNPB). Still other cultures received about 1 mM of dorsomorphin.The cells were incubated in this culture medium for about 3 days. Itwill be appreciated that cells can be incubated about 1-5 days,preferably 2-4 days, and more preferably 3 days.

Similar to the above, methods for determining the composition of thecultures has been previously described in the above relatedapplications, but principally by RNA and protein assays well known inthe art. PDX1-positive foregut endoderm cells express increased levelsof certain signature cell surface markers besides PDX1 such as Sox9,HNF6 and PROX1, but do not appreciably express other markers of laterStage 4 or 5 cells such as or PDX1, NKX6.1, PTF1A, PCA and cMYC found inpancreatic progenitor cells, or NGN3, PAX4, ARX and NKX2.2 expressed inendocrine precursor cells, or INS, GCG, GHRL, SST or PP expressed inpolyhormonal or singly hormonal pancreatic endocrine cells.

Differentiation Conditions for Stage 4

To further promote differentiation of properly differentiatedPDX1/NKX6.1 co-positive pancreatic progenitor cells from PDX1-positiveforegut endoderm cells, the PDX1-positive foregut endoderm cell culturemedium was exchanged and incubated in a medium comprising a similar basemedium as in Stage 3 above, DMEM high glucose/1% Glutamax/1% Pen-Step/1%B27 Supplement, except that there is no RA or retinoic acid derivativesuch as TTNPB or noggin or dorsomorphin. Instead, about 50 ng/mL ofNoggin, KGF and FGF was added to the culture. It will be appreciatedthat about 10 to 100 ng/mL of epidermal and fibroblast growth factors(EGF and FGF) can be added to the culture. There is preferably about 10to 50 ng/mL, or preferably, about 10 ng/mL of EGF and about 50 ng/mL ofFGF added to the cultures. Alternatively no FGF can be added to thecultures, or about 25 to 100 ng/mL each of Noggin, KGF, FGF, orpreferably about 50 ng/mL of Noggin, KGF and FGF was used. The cellswere kept in this medium with media exchanges for about 4 to 5 days. Itwill be appreciated that cells can be kept in medium for about 2 to 6days, preferably 3 to 5 days, and even more preferably 4 to 5 days withpermissible media exchange.

Similar to the above, methods for determining the composition of thecultures has been previously described in the above relatedapplications, but principally by RNA and protein assays well known inthe art. PDX1/NKX6.1 co-positive pancreatic progenitor or endoderm cellsexpress increased levels of certain signature cell surface markers suchas PDX1, NKX6.1, PTF1A, CPA and cMYC, but do not appreciably expressother markers found in later stage cells such as NGN3, PAX4, ARX andNKX2.2 expressed in endocrine precursor cells, or INS, GCG, GHRL, SST orPP expressed in polyhormonal or singly hormonal pancreatic endocrinecells.

Transplantation & Purification of PDX1-Positive Pancreatic Progenitors

After about 3-5 days in the Stage 4 cell culture medium, the cellcultures were either prepared for: i) flow cytometry separation and/orpurification and analysis; ii) encapsulation into cell encapsulationdevices as discussed in more detail above; and/or iii) transplanted intothe mammal. Alternatively, the cell culture from Stage 4 was transferredor adapted in media of DMEM high glucose/1% Glutamax/1% Pen-Step/1% B27Supplement minus the growth factors for about 1 to 2 days, before flowcytometry and/or transplantation.

Detailed descriptions for enriching, separating, isolating and/orpurifying pancreatic progenitors and/or pancreatic endocrine cells orendocrine precursor cells are described in detail in U.S. applicationSer. No. 12/107,020, entitled METHODS FOR PURIFYING ENDODERM ANDPANCREATIC ENDODERM CELLS DERIVED FROM hES CELLS, filed 8 Apr. 2008,which is herein incorporated by reference in its entirety.

Briefly, CD142 was used to enrich for PDX1-positive pancreaticprogenitor (or pancreatic epithelia cells or PE) by quickly washed withPBS and enzymatically dissociated into a substantially single cellsuspension using TrypLE and 3% FBS/PBS/1 mM EDTA (sorting buffer). Thesingle cell suspension was passed through a 40-100 uM filter and thenpelleted and washed again in a sorting buffer, re-pelleted and thenresuspended again as a substantially single cell suspension in sortingbuffer at about 1×10⁸ cells/mL. The resuspended cells were thenincubated with Phycoerythrin conjugated anti-mouse CD142 antibody (BDPHARIMIGEN™) at 10 ul per 1×10⁷ cells. The cells were washed at leastonce with volume sorting buffer, pelleted and resuspended again as asubstantially single cell suspension in sorting buffer containing asolution of anti-Phycoerythrin microbeads (Miltenyi Biotec) andincubated. Cells were washed at least once and immuno-magnetic selectionof CD142-positive cells was performed. The pre-sort, bound and flowthrough fractions were each collected and counter-stained with anti-PDX1and/or anti-CHGA.

The bound fraction was highly enriched for CD142-positive cells and forPDX1-positive pancreatic progenitor cells as compared to the pre-sortedand flow through fractions. See Table 9 of U.S. application Ser. No.12/107,020. For example, the anti-CD142-positive or bound fraction wascomprised of about 71% PDX1-positive pancreatic progenitor cells ascompared to about 22% PDX1-positive pancreatic progenitor cells in thepre-sort fraction and about 8% PDX1-positive pancreatic progenitor cellsin the flow through fraction. Hence, there was about a 3-fold enrichmentin PDX1-positive pancreatic progenitor cells in the anti-CD142-positiveor bound fraction relative to the pre-sort population. Also, theCD142-positive or bound fraction was depleted of chromograninA(CHGA)-positive cells indicating that multi- or singly-hormonalendocrine cells were not selected or enriched in this population. CD142therefore can be used for positive immuno-selection to enrich and/orpurify for PDX1-positive pancreatic progenitors or epithelial cells,whereas the flow through fraction (the fraction or cells not binding tothe antibody column; or CD142-) is enriched with pancreatic endocrinetype cells. Also, refer to Table 10 of U.S. application Ser. No.12/107,020.

Example 5 In Vivo Maturation of Pancreatic Progenitors AmelioratesHypoglycemia in Diabetic Induced Animals

To determine whether the PDX1-positive pancreatic progenitor cellcultures or enriched populations, including the cryopreservedpopulations, were fully capable of developing and maturing in vivo toglucose sensitive insulin secreting cells, the progenitor populationswere loaded into the encapsulating devices similar to that describedabove in Examples 1 and 2 using either a Hamilton syringe with a bluntedappropriately sized gauge needle or centrifuge loading method per themanufacturer's procedure.

Before loading the cells into the device, the device was deemed suitablefor transplantation and use in mammals including humans, e.g., thedevice has passed typical standards of quality control includingsterilization. Because membrane components of the device are likely tobe comprised of hydrophobic membranes, e.g. PTFE and therefore repelwater, sterilizing the devices is typically accomplished by wetting thedevices in an alcohol solvent (e.g. 95% ETOH) and then washing them insaline solution repeatedly. Devices therefore should be kept moist priorto loading. Ideally any device loading method is performed under sterileconditions ensuring that any device component which is implanted willnot be contaminated with unwanted cells.

Device loading can be performed by either using a Hamilton syringe orthe like plus a blunted appropriately sized gauge sterile needle (sizewill vary depending on the diameter of the port of the device) or thelike, e.g., a 22 gauge needle. The needle is connected to a theappropriate Hamilton syringe and contains about 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more μL ofcell volume which reflects a therapeutically effective amount or dose ofcells. The needle is then inserted through at least one a port of thedevice and through to the lumen (or chamber or reservoir) but withouttouching the walls of device. Substantially the entire contents of thesyringe are expelled slowly into the device while at the same time theneedle is being withdrawn.

Alternatively, another method of loading the device using a needle is byusing a sterile plastic or silicone port tube which connects the deviceport to the needle which is inserted into the port but not in the lumen.In this method, a silicone adhesive is injected into the silicone porttube, walling or sealing off the device port. The port tube is then cutoff and inspected for leaks or breaches.

To load the device using a centrifuge method, a certain cell volumecontaining a therapeutically effective amount or dose of cells is drawnup in a micropipette tip and the tip contacted with the device port. Thedevice and the pipette tip can also be put into a larger container orcentrifuge conical tube, either immobilized or not. Often certainvolumes of media is layered on top of the cell suspension in the pipettetip and also in the larger conical tube. The conical tube in thencentrifuge at about 1000 rpms for a few minutes, preferably 20 secondsup to about 2 minutes or until cells are loaded into the device. Thengreat care is used to remove the loading components and secure theloaded device.

The encapsulated cells in the device were then prepared for implantationinto a mammal, e.g., immuno-compromised mice such as SCID/Bg, rat,larger mammal or human patient. Methods of implanting the encapsulatedcells and device is substantially as that described above in Examples 1and 2 and Kroon et al., 2008, except in Kroon et al. the cells areimplanted on a GELFOAM and not contained inside a device. However,because the encapsulated cell population contains substantially aprogenitor population similar to that described by Kroon et al. 2008 andU.S. Pat. No. 7,534,608, titled METHODS OF PRODUCING PANCREATICHORMONES, filed Jul. 5, 2007, which is herein incorporated by referencein its entirety, assays for determining cell functionality weresubstantially the same. Briefly, the animals were tested about everytwo, three or four weeks by injecting them with a bolus of arginine orglucose, preferably glucose, which if the encapsulated cells haveproperly matured into now beta cells in vivo, will secrete insulin inresponse to the glucose. In short, the mature beta cells are responsiveto glucose not unlike naturally occurring beta cells. Blood wascollected from the mammal to determine levels of human C-peptide whichis secreted from the human transplanted progenitor cells having maturedinto human beta cells. Human C-peptide was detected in animal serum asearly as 4 to 6 weeks after transplantation and the levels of humanC-peptide increase over time as more progenitors or endocrine precursorcells mature into properly functioning beta cells. Typically amounts ofhuman C-peptide above 50 pM were considered an indication of function ofthe transplanted cells. It was previously shown that engrafted cellsfrom the PDX1-positive pancreatic progenitors faithfully give rise toendocrine cells expressing markers and physiological characteristic offunctioning pancreatic hormone-secreting cells. See Kroon et al. 2009,supra and U.S. application Ser. No. 11/773,944, titled METHODS OFPRODUCING PANCREATIC HORMONES filed Jul. 5, 2007, which is hereinincorporated by reference in its entirety.

Immuno-suppression is contemplated for certain mammals for an initialinterim period until the progenitors inside the device fully mature andare responsive to glucose. In some mammals immuno-suppression regimensmay be for about 1, 2, 3, 4, 5, 6 or more weeks, and likely depend onthe mammal.

Lastly, similar to Kroon et al. 2008, the encapsulated cells not onlymatured into pancreatic islet clusters with endocrine cells but alsodeveloped into islet associated cells such as acinar cells. Thus, thetransplanted PDX1-positive pancreatic progenitors were not committed tobecoming just singly-hormonal endocrine secreting cells but were capableof maturing and developing into what is substantially similar to a humanislet, comprising both endocrine and acinar cells. And this in vivomaturation and glucose responsiveness of the transplanted cells wasobserved whether the progenitor cells (PDX1/NKX6.1 co-positive;endocrine precursors, or certain poly-hormonal or singly-hormonal cells)were cultured and differentiated in vitro and subsequently transplanted,or whether certain progenitors were purified or enriched beforetransplantation, or whether they were previously made from one or morebatches and cryopreserved, thawed and adapted in culture beforetransplantation.

Briefly, after transplant, the transplanted cells were allowed todifferentiate and further mature in vivo. To determine whether thetransplanted cells had normal physiological function as a naturallyoccurring beta cell for example, levels of human insulin were determinedby testing levels of human C-peptide. Human C-peptide is cleaved orprocessed from human pro-insulin, hence, the detection of humanC-peptide, and not endogenous mouse C-peptide, indicates that insulinsecretion is derived from the grafted (exogenous) cells.

Glucose stimulated human C-peptide secretion of the transplanted cellsin serum was measured at various time points post transplant. It will beappreciated that glucose stimulated human C-peptide secretion can bemeasured at various time points, e.g. at least 30, 35, 40, 45, 50, 55,60, 65 and more days. Glucose stimulated human C-peptide levels could beacutely measured in the serum as early as about 15 minutes post-glucoseadministration or injection. Blood was withdrawn from the animals atabout 15, 30 and 60 minutes time intervals post glucose administration.The serum was separated from the blood cells through centrifugation inmicro-containers as described by the manufacturer (Becton Dickinson).The ELISA analysis was performed of the serum using ultrasensitive humanspecific C-peptide ELISA plates (Alpco). In general, more than themajority of animals receiving the encapsulated transplanted cellsresponded to glucose as demonstrated by levels greater than thresholdlevels 50 pM of human C-peptide.

In summary, wholly encapsulated cells by the above device does notaffect maturation of the cells nor the physiological function of thecells once they have matured. Further, the amelioration of hypoglycemiain these diabetic induced animals was observed and was substantiallysimilar to that previously described in Kroon et al. (2008) supra, aswell as in U.S. Pat. No. 7,534,608, although neither described whollyencapsulated transplanted cells or grafts. These references are hereinincorporated by reference in their entireties.

Accordingly, it will be apparent to one skilled in the art that varyingsubstitutions, modifications or optimization, or combinations may bemade to the embodiments disclosed herein without departing from thescope and spirit of the invention.

All publications and patents mentioned in this specification are hereinincorporated in their entireties by reference.

As used in the claims below and throughout this disclosure, by thephrase “consisting essentially of” is meant including any elementslisted after the phrase, and limited to other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

1.-20. (canceled)
 21. A method for implanting cells into a hostcomprising: a. implanting a device into the host; b. allowing the deviceto vascularize; and c. loading the device with a human embryonic stem(hES) cell-derived pancreatic progenitor cell population, therebyimplanting cells into a host.
 22. The method of claim 1, wherein the hEScell-derived pancreatic progenitor cell population comprises pancreaticprogenitor cell aggregates expressing pancreatic and duodenal homeoboxgene 1 (PDX1).
 23. The method of claim 1, wherein the host isimmuno-suppressed.
 24. The method of claim 1, wherein the device is notpermeable to immune system cells of the host.
 25. The method of claim 1,wherein the hES cell-derived pancreatic progenitor cell population iswholly encapsulated.
 26. The method of claim 1, wherein the devicecomprises a cell encapsulation chamber and at least one seal is withinthe cell encapsulation chamber wherein said seal within the cellencapsulation chamber does not increase the surface area of the chamber.27. The method of claim 1, wherein the device comprises more than onecell encapsulation chamber.
 28. The method of claim 1, wherein the hEScell-derived pancreatic progenitor cell population is angiogenic. 29.The method of claim 1, wherein the device is vascularized on theexterior and interior of the device.
 30. The method of claim 1, whereinthe hES cell-derived pancreatic progenitor cell population matures invivo and produces insulin.
 31. A method for implanting cells into a hostcomprising: a. implanting a housing into the host; b. allowing thehousing to vascularize; c. removing the housing; and d. implanting adevice into the host comprising a human embryonic stem (hES)cell-derived pancreatic progenitor cell population, thereby implantingcells into the host.
 32. The method of claim 31, wherein the hEScell-derived pancreatic progenitor cell population matures in vivo andproduces insulin.
 33. The method of claim 31, wherein the housingcomprises an outer sleeve.
 34. The method of claim 33, wherein thesleeve is not removed prior to step c.
 35. The method of claim 31,wherein the hES cell-derived pancreatic progenitor cell populationcomprises pancreatic progenitor cell aggregates expressing pancreaticand duodenal homeobox gene 1 (PDX1).
 36. The method of claim 31, whereinthe host is immuno-suppressed.
 37. The method of claim 31, wherein thedevice comprises a cell encapsulation chamber and at least one seal iswithin the cell encapsulation chamber wherein said seal within the cellencapsulation chamber does not increase the surface area of the chamber.38. The method of claim 31, wherein the device comprises more than onecell encapsulation chamber.
 39. The method of claim 31, wherein thedevice is refillable.
 40. The method of claim 31, wherein the device isvascularized on the exterior and interior of the device.